Use of tree sap to preserve sperm cell lines

ABSTRACT

A method of cryogenically preserving sperm comprising (a) combining sperm to be cryogenically preserved and a composition that comprises (1) a cryoprotectant, comprising one or more tree saps; and (2) an extender medium to produce a sperm/medium combination; and (b) subjecting the combination to conditions that result in cryopreservation of sperm, thereby producing a cryopreserved combination that comprises cryopreserved sperm is disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 62/153,197, filed Apr.27, 2015 and incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

N/A

BACKGROUND

In general, the present invention involves the use of tree sap tocryogenically preserve avian and mammalian sperm cells, preferably foruse in the poultry industry, birds of prey preservation, andpreservation of endangered or threatened avian species. The presentinvention may also be used in the cattle industry, pig industry, equineindustry, and in mammalian veterinary medicine.

Avian spermatozoa have a shape that makes the spermatozoa hard tofreeze. The spermatozoa are long and thin and are shaped like a whip.This makes the cells very subject to cryogenic injury because they havea large surface area that can be damaged easily upon freezing orprocessing. Mammalian sperm will also benefit from the present inventionbecause even though these cells are easier to freeze, they are stillsubject to damage from the cryogenic processes. (Reference; Avian SemenCryopreservation: What Are the Biological Challenges? J. A. Long, 2006Biotechnology and Germplasm Laboratory, Animal and Natural ResourcesInstitute, Beltsville Agricultural Research Center, AgriculturalResearch Service, USDA, Beltsville, Md. 20705, 2006 Poultry ScienceAssociation, Inc. Accepted Sep. 10, 2005)

Currently, avian spermatozoa are frozen using several techniques. Onetechnique uses the addition of a cryoprotectant to a fluid media thatsuspends and supports the cells. The first step in the procedure is tocollect the semen and then add a liquid extender. A semen extender is aliquid diluent which is added to semen to preserve its fertilizingability. The extender allows the semen to be freighted to the female,rather than requiring the male and female to be near to each other.Special freezing extender also allows cryogenic preservation of sperm(“frozen semen”), which may be transported for use, or used on-site at alater date.

This extender/cell mixture is then placed in a refrigerator to chill themixture down to a desired temperature that allows the cells to line upthe lipid components in their outer cell membrane prior to freezing.This is a form of “cold acclimation” and helps to allow the cells tosurvive the cryogenic process. The method also reduces the temperaturegradient drop that the cells have to go through before they reach thefreezing point and reduces the cell damage when being frozen.

Once the cells are chilled/acclimated, the cryoprotectant is added tothe extender/cell mix, the mixture is packaged quickly and either flashfrozen by quick immersion in the liquid nitrogen, pelletized and flashfrozen and then packaged into cryo-vials, or suspended above the liquidnitrogen in the vapors to freeze more slowly before it is immersed inthe liquid nitrogen. Both fast and slow freezing can be done based onspecies requirements. Different cryoprotectants that are added to themix commonly include DMSO (Dimethyl sulfoxide), MA (Methyl-Acetamide),and DMA (Dimethyl Acetamide). These chemicals act as intracellularcryoprotectants while the non-cell wall-permeable chemicals act asextracellular cryoprotectants. These are also known to damage the cellwall during cryopreservation and this impairs fertility.

A better and more effective way of preserving avian and mammalian semenis needed in the art.

SUMMARY OF THE INVENTION

In one embodiment, the present invention is a method of cryogenicallypreserving sperm comprising (a). combining sperm to be cryogenicallypreserved and a composition that comprises (1) a cryoprotectant,comprising one or more tree saps; and (2) an extender medium to producea sperm/medium combination and (b). subjecting the combination toconditions that result in cryopreservation of sperm, thereby producing acryopreserved combination that comprises cryopreserved sperm. In oneversion of the invention the sperm is avian sperm. In one version thesperm is derived from the Northern goshawk (Accipiter gentilis).

In another version of the invention the sperm is derived from amammalian non-human species, preferably selected from the groupconsisting of cattle, pigs and equines.

In one version of the invention the sap is either maple tree sap orbirch tree, preferably both first run saps.

In one version, the present invention is the cryopreserved combinationresulting from the method described above.

In another version, the present invention is a method of fertilizing anegg cell comprising the step of introducing the combination describedabove to an unfertilized egg cell, wherein the egg becomes fertilized.

DESCRIPTION OF THE INVENTION

In general, the present invention is a method and medium useful for thecryogenic preservation of sperm using tree saps. In another embodiment,the present invention is a composition comprising the mixture of thepreservation medium and the sperm, using tree saps.

Although the present invention is useful for all animal sperm, theinvention is most preferably used with avian sperm because of thespecial physiologic needs of the avian samples. Preferred avian speciesinclude birds of prey (such as Falconiforms and Strigiformes) andcommercial species such as turkeys, chickens (Galliformes) and ducks(Anseriformes.) Other preferred avian species include but are notlimited to Passeriformes and Psittacifomes.

In another version of the invention, one may wish to preserve the spermof other mammalian species, including cattle (Family—Bovidae), pigs(Family—Suidae), horses (Family—Equidae) and veterinary medicineapplications, including canine (Canidae) and feline (Felidae) species.

In certain embodiments, the method of cryogenically preserving spermcomprises: (a) combining sperm to be cryogenically preserved with amedium comprising (1) a cryoprotectant, such as one or more tree saps orits extracts; and (2) an extender designed to support cell life, whereinthe combination produces a sperm/medium combination (cryoprotectivemedium/sperm combination); and (b) subjecting the combination toconditions that result in cryopreservation of sperm, thereby producing acryopreserved combination that comprises cryopreserved sperm.

The typical sperm extender typically contains chemicals to bothstabilize and protect cell membranes. The Examples below use BeltsvilleTurkey Extender (BTE) recipe with the exception of removing fructose asone of the ingredients. The fructose was replaced with sucrose andconstitutes a separate extender recipe, also a preferred embodiment ofthe present invention. It was found that goshawk semen did not do wellwith fructose as its energy source when being cryogenically preserved.This observation is true for other animal cell lines.

The preferred extender recipe for goshawk semen, and other typical aviansemen samples, consists of;

Potassium Diphosphate 3H2O 12.7 gramsSodium glutamate 8.675 gramsSucrose to replace Fructose (Anhydrous) 5.000 gramsSodium Acetate 3 H2O 4.255 gramsTES 1.95 grams

N-tris Hydroxymethyl Methyl-2Amino-ethane Sulfonic Acid

Potassium citrate 0.64 gramsPotassium Monophosphate 0.65 gramsMagnesium Chloride 0.338 gramsPurified water 1022 ml is added to the dry ingredients.

This constitutes a full recipe of the preferred extender for goshawksemen for cryopreservation. The sucrose is often left out of this recipeand supplied just through the addition of tree saps that naturally havesucrose in them. Other base recipes may be preferred for other celllines in other species to meet those species specific requirements. Thesugar supplied for the recipe may come from the sap as in the Exampleslist in the Excel Spreadsheet.

The present invention involves the use of tree sap as a cryoprotectant.Tree sap is a fluid transported in xylem cells (tracheids or vesselelements) or phloem sieve tube elements of a plant. Two kinds of sap aredefined as either Xylem sap or Phloem sap. We include both kinds of sapin our definition.

Tree sap is produced at a time of the year when the trees are goingthrough cold stress and freezing in the temperature ranges that are mostharmful to the cells that we are trying to freeze. The trees survivetemperatures from freezing to minus 60° F. The trees also survive thedaily shift in temperatures that the tree can survive both well abovefreezing to well below it. The tree sap contains properties that allowit to support cell life even when frozen and when going through rigorousfreeze thaw cycles and daily temperature extremes. It contains varioussugars, antifreeze proteins, carbohydrates, minerals, phenoliccompounds, and other compounds that provide cryoprotective properties.Some of these compounds have yet to be described.

The tree species that are most useful in this invention includes thecold-hardy maple tree species, birch tree species, poplar tree species,aspen tree species, and other trees that can be tapped or wherechemicals or fluids can be extracted from them. Tree species from thehigher latitude deciduous forests are included even if not listeddirectly herein.

A common factor in these trees is the amount of sugar in the sap. Sugarshave cryoprotective properties. Some avian extender recipes often callfor 0.5% of either sucrose or fructose. Most tree species meet or exceedthis percentage sugar requirement. Maple tree range anywhere fromapproximately 0.5% to 4.0% sucrose. Another common factor that thesetrees have is that they have non-sugar cryoprotectant chemicals in theirsap. These chemical may provide stronger cryoprotective properties thanthat the simple sugars that are easily measured.

There are over 128 species of maple trees worldwide. The sugar maple(Acer saccharum) and black maple (Acer nigrum) produce the most sugar intheir saps. The red maple (Acer rubrum) and the silver maple (Acersaccharinum) produce less sugar but are in the latitudes where they willlikely contain similar cryoprotective properties in their sap. Theselater two species are one preferred version of the present invention dueto the lower sugar content and potentially higher cryoprotectiveproperties in the sap that are not from sugars.

Birch tree (Family—Betulaceae, Genus—Betula), poplar tree (FamilySalicaceae, Genus—Populus), and aspen tree (Family—Salicaceae,Genus—Populus) species come from higher latitudes in the United Statesand Canada and have lower sugar content in their sap than the maple(Acer species) tree species do. The non-sugar cryoprotective chemicalsin their sap will likely be higher as these species survive in a moreextreme environment with temperature ranges fluxuating widely belowfreezing, and the trees have a lower content of the sugars that areknown to be cryoprotective in their sap.

Twenty three species of trees that can be tapped in the United Statesand are useful in the present invention include but are not limited toSugar Maple (Acer saccharum), Black Maple (Acer nigrum), Red Maple (Acerrubrum), Silver Maple (Acer saccharinum), Norway Maple (Acerplatanoides), Boxelder (Acer negundo), Bigleaf Maple (Acermacrophyllum), Canyon Maple or Big Tooth Maple (Acer grandidentatum),Rocky Mountain Maple (Acer glabrum), Gorosoe (Acer mono), Butternut orWhite Walnut (Juglans cinerea), Black Walnut (Juglans nigra), Heartnut(Juglans ailantifolia), English walnut (Juglans regia), Paper Birch(Betula papyrifera), Yellow Birch (Betula alleghaniensis), Black Birch(Betula lenta), River Birch (Betula nigra), Gray Birch (Betulapopulifolia), European White Birch (Betula pendula), Sycamore (Platanusoccidentalis), Acer ginnala, and Ironwood or hophornbeam (Ostryavirginiana).

Preferably, one would begin with extender recipes designed for thepreservation or storage of animal sperm. Typically, the initial amountof sap added to the modified extender recipe will make the initialsolution physiologically close to the osmolality of the raw semen andstill provide for cryo-protection of the cells. The initial osmolalityrange needed is determined by the measurement of the osmolality of theraw semen.

Goshawk semen has an osmolality of about 341 mili-osmoles. Lateradditions of extender/sap combinations increase the osmolality of themixture to dehydrate the cells immediately prior to freezing.Dehydrating the cells just prior to freezing them, increases survival.

An ideal osmolality level is determined by the end results of thesurvival of the cells in question and the ability of the stored sampleto create fertile female gametocytes. It is known that different speciesof birds have sperm cells that tolerate different osmolality extremes.Some avian spermatozoa survive very high osmolality and others do not.[See Species Variation in Osmotic, Cryoprotectant, and Cooling RateTolerance in Poultry, Eagle, and Peregrine Falcon Spermatozoa; Juan M.Blanco, George Gee, David E. Wildt, and Ann M. Donoghue; Biology ofReproduction Oct. 1, 2000 vol. 63 no. 4 1164-1171] The use of the sapallows both the removal of other toxic cryoprotectants from the mixand/or reduces the amount of toxic cryoprotectants used. Yet, one maystill wish to add additional cryoprotectants to the mix.

A typical sperm/sap-extender combination of the present invention is asfollows: The final volume of the sperm/sap-extender combination shouldbe no more than 1:3 dilutions making the semen a quarter of the finalvolume. Semen dilutions higher than this can impair fertility because ofsimple dilution.

In a preferred version of the invention, sap comprises at least 30% ofthe final sperm/extender combination. In another version of theinvention, sap comprises 10%-80% of the final sperm/extendercombination, preferably about 50%.

Over dilution reduces sperm fertility of the sample. The amount of sapneeded to provide cryogenic protection to the mixture variesconsiderably because of tree species variation in cryoprotectant typesand properties.

I have been modifying the extender recipe enough to allow sap to beblended into the mix so that this mix then supports the cells when theyare frozen in liquid nitrogen (LN₂) with or without the use of anadditional cryoprotectant. A typical example of extender includes thedry ingredients of the Beltsville Turkey Extender without the fructose(BTE minus fructose) as a base recipe to work with.

The sap from the Maple tree and the Birch tree were then added to theBTE minus fructose and used at different ratios, to preserve the spermcells. A recipe that preserved the cells in LN₂ well included 1 part rawsemen, 1 part BTE minus fructose with 0.5% sucrose added back in, 2parts BTE minus fructose with sap added, to supply its liquid volume.The sperm and the BTE minus fructose with 0.5% sucrose added back in;were mixed in a 0.5 ml Eppendorf vial in the fridge. A matching volumeof BTE minus fructose with sap as its liquid diluent was also placed inthe fridge but in a separate tube. Both tubes were allowed toequilibrate to an equal temperature for 10 minutes before they were thenmixed, packaged, and then flash frozen. The work was done in the fridgeat 42° F. so there were no temperature fluxuations to stress the semen.This form of cold “acclimation” allowed the lipid component of the cellwall to line up prior to freezing to help prevent damage to the cellstructure.

Therefore, a preferred version of the present invention comprises acomposition, wherein 1 part of the volume is raw semen; 1 part of thevolume is extender, such as BTE no fructose plus 0.5% sucrose added backin; and 2 parts of the total volume was BTE no fructose with sap ofeither the maple or birch tree. A minimum of 50% sap by volume shouldpreferably be used in this mix. Samples with 50% sap by volume had farbetter survival on thaw than samples with less than this percentage.

The packaging consisted of the semen being placed in a 75 ul Mylarcoated capillary tube with one end being caulked. Its opposite end wasleft open. This capillary tube was then placed inside a standard plasticpoultry straw and the end opposite of the cotton was crimped shut. Thepoultry straw was then placed inside a plastic soda straw that had holescut in the side of it. These holes allowed the LN₂ to enter and surroundthe poultry straw quickly as it was immersed. The holes in the sodastraw also allowed the package to drain and breathe as it was thawed sothat it did not explode.

Cryopreservation can be carried out at any time after production of themedium/sperm combination as long as the storage does not significantlyadversely affect the viability of the sperm. For example,cryopreservation can often be carried out as long as 180 minutes afterthe sperm/medium combination is produced with no loss of fertility.Samples should be chilling to extend the shelf life before freezing. Atypical temperature for storing avian semen at is 5° C. Chilling thesemen helps to line up the lipid component in the cell wall prior tofreezing. This increases cell survival.

Preservation is typically carried out at a temperature minus −198° F. Inspecific embodiments, cryopreservation is carried out at a temperaturebetween from about minus −80° F. to about minus −198° F. In onepreferred embodiment, the cryo-protection takes place in a liquidnitrogen bath/canister and the vials are stored at a −198° F. Long termstorage can be achieved by placing the storage vials or straws in aliquid nitrogen canister. One would then wish to use the preserved spermto fertilize a female gametocyte, female germ cell or ovum.

Before the sperm is used for artificial insemination or incubated with afemale gamete, the sperm is typically thawed and may also be washed.Sperm samples are often thawed in cold water or warm water baths withthe temperature requirements being determined both by the species cellrequirements or the cryoprotectant type used in the mix. Avianspermatozoa are typically thawed in ice water baths or cool water baths,and bovine spermatozoa are typically thawed in warm water baths that arebody temperature. Insemination is performed immediately after thaw.Sperm are sometimes concentrated into pellets with the contents ofdifferent straws being combined, centrifuged down, to form a pellet ofsemen.

In all embodiments described herein, the resulting cryopreserved spermcan be stored indefinitely.

The fertilization capacity or ability of sperm can be assessed usingmethods known to those of skill in the art, such as in vitro methods,including assessing the ability to fertilize the oocytes/female gametewith which they are combined/incubated (their ability to form-cellembryos, for example) and/or in vivo methods, including assessing theproduction of offspring by females into whom the fertilizedoocytes/female gamete are implanted (mammals). Fertilization capacity orability can be assessed using available methods, such as a functionalassay, including, but not limited to, a motility assay, a viabilityassay, a hemizona assay (binding of the sperm to the zona pellucida) orsperm penetration into zona-free mammalian or avian oocytes.

The commercialization of cryogenically freezing avian semen has eludedscientists for decades. The freezing process has not been successfulenough. Current papers cite approximately 35 to 40% semen motility afterthaw. See; Comparative cryopreservation of avian spermatozoa; Benefitsof non-permeating osmoprotectants and ATP on turkey and crane spermcryosurvival. By Juan M. Blanco, Julie Long, George Gee, David E. Wildt,Ann M. Donoghue, Received 24 May 2010 Accepted 10 Dec. 2010. ElsevierB.V.

The present invention, comprising the improvement of using sap as thesole cryoprotectant, often showed greater than 50% survival based onLive/Dead stains done after the thaw of samples. Some examples showed upto 73% survival on thaw with no additional cryoprotectant being used.Once this successful idea is combined with the other currentlysuccessful ideas of science, the survival of the semen will likely behigh enough to make avian semen cryopreservation a commercially viableventure.

Other animal species will likely benefit from this invention as well.There are numerous articles on scientists trying to freeze the semen ofother animal species with limited success. The use of tree sap harvestedat winter's first thaw, and used in cryopreservation of cell lines is anexciting and now documented success. The success of this process mustalso be evaluated based on the improved fertility and hatchability ofeggs produced from females inseminated with frozen semen.

EXAMPLES

In general, the present invention involves the use of tree sap tocryogenically preserve avian sperm lines, preferably for use in thepoultry industry, birds of prey preservation, preservation of endangeredor threatened avian species, and other avian species. It will also beuseful in pigs (Family—Suidae), cattle (Family—Bovidae), horses(Family—Equidae), dogs (Family—Canidae), and cats (Family—Felidae).

Table 1 contains the results of many experimental trials. In general, Iobtained maple tree sap from the native trees in southeastern Wisconsin.The osmolality of Maple tree #3 is 100 mili-osmoles.

The raw dry ingredients for the preferred medium were as follows:Beltsville Turkey Extender recipe, minus the fructose; had maple tree orbirch tree sap added for a final volume of 100 ml. (I added 90 ml of sapto make the final volume of the dry and wet ingredients total 100 ml.)

I began with a set of dry ingredients that was for 1/10^(th) of thestandard recipe listed above. I added 90 ml of maple tree sap to one jarand 90 ml of birch tree sap to another jar to make a final volume of 100ml in each jar. No other cryoprotectant was added into the mix. Thefructose had been removed so the energy source for the semen came fromthe sucrose that was already in the sap. The cells that I am trying topreserve do not appear to metabolize fructose well and need the sucrosein the recipe to survive the freezing.

The sap was used full strength in the stock jars, but it was used indifferent ratios when it was added to raw semen. Sometimes a 1:1:2dilution was used (1 part semen: 1 part BTE no fructose, plus 1/%sucrose: 2 parts BTE plus Sap); sometimes a 1:1:1 dilution was used.Sometimes a 1:1:1 dilution was used where the final mix was 33% Semenand 66% sap with sap being added into the both the base mixture and thefinal mixture before freezing. In all cases no other cryoprotectant wasadded to the sample and only the sap was used to preserve the cells inthe liquid nitrogen. The cells survived in large percentages even whenno additional (penetrating or non-penetrating) cryoprotectant was addedto the mix.

Experiments were also done using the sap from Alaskan birch tree. Again,the raw dry ingredients for the Beltsville turkey extender, minus thefructose ( 1/10^(th) volume of the standard recipe) had birch tree sapadded for a final volume of 100 ml. [The recipe for a standard litervolume of BTE is listed above.] No other cryoprotectant was added intothe mix.

The maple tree sap had been stored in 100 ml plastic bottles, with about16 bottles per cardboard box, with the top left open. The top of thebottle had the minerals and other chemicals forced out of it leaving theice crystals at the top. The center of the bottle had not frozen, and itremained in an almost glass like state without freezing completely after24 hours. This type of freezing is critical to success when doingcryogenic freezing. This prevents ice crystal formation that damages thecells. [Reference; Investigation of Chemical and Physical Properties ofSouthwestern Wisconsin Maple Syrup; By Hiroyuki Takano, A ThesisSubmitted in Partial Fulfillment of the Requirements for the Master ofScience Degree with a major in Food and Nutritional Sciences. Martin G.Ondrus, Thesis Adviser; the Graduate School University ofWisconsin-Stout, December 2005]

The sap of the birch tree was obtained from Alaska through a syrupcompany called Alaska Wild Harvest LLC, dba Kahiltna Birchworks, PO Box2267, Palmer, Ak. 99645. I obtained both the first run and second runsaps for experimentation. This sap contains three times less sugar onaverage, than Maple tree sap. This tree comes from higher latitudes thatare subject to more severe temperatures and temperature swings than theforest in Wisconsin are. The birch tree sap froze very slowly in thechest freezer and in a similar manner to that of the maple tree saplisted in the previous paragraph.

The first semen sample that I froze was from a male Northern goshawk(Accipiter gentilis) using an extender recipe that was modified toinclude maple tree sap. This recipe consisted of all of the dryingredients of the Beltsville Turkey Extender in the usual percentages,without the fructose. 90 ml of maple tree sap was added for a finalvolume of 100 ml. (This is 1/10^(th) of a standard recipe for BTE) Onehundred percent of the liquid added into the recipe was maple tree sap.The sucrose content of the maple tree sap is reported in the literatureto be between 2%-2.6%. The exact sucrose level of this sap was notmeasured but estimated to be about 2% because this was a first run sap.The needed minimum sugar level for the Beltsville Turkey Extender is0.5%. So this recipe ended up having more sugar in it (than thecommercial extender) because the sap had 4-5 times the needed sugarlevel, naturally in its sap.

This first recipe contained 4-5 times the needed sugar, making ithyperosmolar so that the sperm would gradually lose motility at roomtemperature. However, a frozen semen sample (Sample #69, Table 1) fromthe Northern goshawk (Accipiter gentilis) was immediately flash frozenupon mixing with the extender-sap combination 1:2 (1 part semen and 2parts Extender/Sap combination) and 58% of the cells survived thefreezing and thawing process based on a live/dead stain (Eosin/Nigrosin)and visual observations. This sample was thawed in a cool water bath atapproximately 55° F. after being in the liquid nitrogen can for over aday. These cells then went on to lose motility at nearly at exactly thesame rate as a sample that had been mixed and held at room temperaturedue to the chemical makeup of the sample. However, the cells survivedthe freezing process essentially unchanged. The motility and linearmovement of the cells was left nearly intact, being unaltered by thefreezing process. This was my first documented success and it exceededmy expectations. Cell survival post freezing showed great success.

There was no other cryoprotectant put into the sample. This recipe isclearly hyperosmolar (and detrimental to the cells) because it containedat least 2% sucrose and the sperm only needed 0.5% sucrose.

I found 58% survival upon thaw based on a live/dead Eosin/Nigrosin stainon this first sample. A hundred cells were counted using a standard labcell counter and this simple percentage established. This exceededliterature references of 25% with standard cryoprotectants such as DMAand MA. A survival rate of above 25%, preferable above 40% or 50%,indicates a successful experiment.

A second sample of 22 ul semen (sample #81, Table 1) was then frozen. 22ul Beltsville Turkey Extender, no fructose, plus 0.5% sucrose was addedto the semen in a 0.5 ml Eppendorf tube and placed in the fridge. 44 ulof BTE with maple tree sap; was placed in its separate tube, also in thefridge at 42° F. The two liquids were combined after acclimating in thefridge for 10 minutes. The total volume was 88 ul. The sample waspackaged in 2-75 ul Mylar coated capillary tubes, placed in poultrystraws, this was then placed in a ventilated soda straws, and flashfrozen. Both straws were thaw in a 55° F. water bath. Two straws wereproduced from one semen sample. Sample 1 had 25-30% live forwardlymotile sperm with normal speed of travel and a live/dead stain of 50live/50 dead. The second straw had 55% forwardly motile with normalmotility and a live/dead stain of 57 live/43 dead. The semen survivalincreased when the percentage of the Maple sap was lowered to 50%.

Additional samples of semen from this male goshawk were frozen. Theresults are listed in Table 1, a table disclosing semen samples whereeither maple sap or birch sap were used exclusively forcryopreservation. The semen of the Northern goshawk (Accipiter gentilis)was used in all experiments.

I list samples in Table 1 that are successful and those that are not inthe column marked “Is this sample workable?” Samples were listed as Yes,No, and Maybe. There is a column that lists the success or lack ofsuccess; so it is easy to review the table quickly by looking down thissingle column. I had success freezing samples in LN₂ as soon as Istarted to add the natural Maple tree sap into the formula. Semen cellssurvived cryogenic freezing when only tree sap was used as thecryoprotectant, even when there was no other chemical cryoprotectantused. My samples survived the trauma of freezing almost as if they hadnever been frozen; continuing to swim at a normal speed in a straightdirection. The cells eventually lost motility due to problems associatedwith the solution that they were put in.

It is of particular note that some of the samples survived with evenhigher survival percentages and motility without the addition of othercryoprotectants, where only the sap was used. Sample 84 survived thebest and is nearing the noted highest percentage of survival andmotility known to scientists that work in this field of cryopreservationat 73L/27D % live/dead stain and a second sample with a 64L/36D %live/dead stain. Samples 67, 69, 70, 71, 72, 80, 81, 84, 93, 97, 118,120, 126, and 128 show very encouraging results with progressive forwardmotility and the live dead stain percentages listed in the chart above.Other samples also showing this trend are also listed in the tablebelow.

Percentage Survival on Live/Dead Stain Sample numbers in Table 1  0-4%Survival 106, 113, 116, 117  5-9% Survival 82, 100, 101, 105, 109, 110,115, 116 10-14% Survival 82, 88, 95, 105, 107, 110, 111, 111, 113, 11415-19% Survival 87, 88, 93, 100, 107, 108, 109, 115 20-24% Survival 82,91, 100, 102, 103, 106, 108 25-29% Survival 99, 99, 114, 116, 117 30-34%Survival 72, 77, 83, 89, 90, 90, 102, 109, 109, 119, 119 35-39% Survival44, 77, 77, 85, 91, 92, 99, 111 40-44% Survival 68, 85, 80 45-49%Survival 72, 83, 118 50-54% Survival 81, 97, 103, 105, 107, 110, 11255-59% Survival 69, 80, 81, 93, 93, 128 60-64% Survival 84, 120 65-69%Survival 72, 76 70-74% Survival 84

Some of the samples showed quiescense features and according to alive/dead stain, survived the freezing but were not motile. Live cellsdo not take up Live/Dead stain and show up as white on microscopeslides, even when no longer motile. These samples are likely not deadand can be“resurrected” and made motile with known techniques. Many ofthe samples had cells with near normal gross cellular features postfreezing and did not appear to be distorted or damaged from the freezingprocess; on the live/dead stains. These stains have been retained forfuture reference.

The sap is the key ingredient for cryopreservation because it isnon-toxic, has no contagious agents to transmit to the sperm, isplentiful, has key cryo-preservative properties, is in a liquid statenaturally, can be collected without bacterial contamination, and it isnot viscous (thick) so it does not impair spermatic motility through thefluid medium. It is a natural product that is very unlikely to containadulterant chemicals.

I envision typical optimizations of the present invention. First, theoptimized-liquid base that supports the cell lines needing to bepreserved will need to be developed and then modified to allow theaddition of the sap to the mix in various percentages, so that the sapdoes not add chemicals in concentrations that would then kill the cells,but would still allow for cryo-protection. (For example, the osmolalityof maple sap that I obtained was 100 mili-osmoles.) This osmolalityappeared to be too high when it was added directly to Beltsville TurkeyRecipe dry ingredients that did not have the fructose added into therecipe. The cells survived the freezing in great shape, but lostmotility possibly due to the hyperosmolality of the approximately 2%sucrose in the maple sap.)

Second, the sap ingredient may be optimized just by choosing differentspecies of trees to use. The sugar content in the saps varies with thetree species and so do the other chemicals that act as naturalcryoprotectants that are not sugars. Syrup producers use maple treesthat produce the most sugar and some syrup producers use birch trees forthis process. They know that where maple tree sap is boiled down,between 20-50 units per one unit of syrup is required. When birch treesap is boiled down, 150 units per 1 unit of syrup is required. Syrupproducers do not tend to use maple tree species that produce low sugarcontent in their sap. Yet, these trees also survive the rigoroustemperatures and temperature extremes and must be adapted well tosurvive without sugar as a main cryoprotectant, implying that otherchemicals in the sap that are not sugars, are acting in this manner.

Additionally, one might wish to use a combination of saps. Combinationsof the maple and birch sap recipes were used in experimentation listedin Table 1. High success rates were achieved with this combination.

Additionally, sap taken at different times during the tapping processmay yield some beneficial results. Later run saps are lower in thesugars seen in the earlier run, while the trees are still going throughand surviving extreme low temperature stresses. The osmolality of thesaps taken at different times may be of benefit.

Additionally, extracts of the saps may yield benefits through thediscovery of newly discovered antifreeze proteins or compounds thatwould be of use with this process. [Reference; When plant cells cansurvive ultra-low temperatures; Pawl M. Pukacki, Physiology of AbioticStress Laboratory, Institute of Dendrology, Polish Academy of Sciences,Kornik, Poland].

Suitable Extender Recipes for 2015 and Semen Survival Study.

Number 1 Recipe;

Beltsville Turkey Extender, No fructose plus 90 ml Maple Tree sap, fromfirst run tapping, QS to 100 ml. Dated on bottle Mar. 11, 2015, mixed onMar. 27, 2015, Initial pH listed as 7.5 and then after being mixed thepH was 6.73 with my meter.

Maple tree sap is 2-2.6% sucrose which is 4-5 times too high andhyperosmolar. BTE normally has 0.5% Fructose in it. However goshawk eggsdo not do well with Fructose and must have Sucrose to survive.

Number 2 Recipe;

Beltsville Turkey Extender, No fructose plus 90 ml Birch tree sap fromAlaska, first run sap, QS to 100 ml. dated on bottle Mar. 11, 2015 andmixed on Apr. 11, 2015. Initial pH listed as 7.5 and then after mixingread on my meter as 7.62.

Number 3 Recipe;

Beltsville Turkey Extender, No fructose plus 0.5% sucrose, plus 16%Methyl Acetamide by weight. The pH was 7.78. Plus 0.2 mg Inositol(should have been 0.02 mg Inositol). It had a total volume of about 10ml.

Number 4 Recipe;

Beltsville Turkey Extender, No fructose, plus ½% sucrose. The final pHwas 7.36. The water was boiled and probably has a low oxygen tension.

Purdy formulas were a simple addition of maple tree sap, by volume toBTE. The osmolalities were as listed.

BTE, Control, No sap added, 352 mOsm

BTE, 5% Maple tree sap, 339 mOsm

BTE, 10% Maple tree sap, 326 mOsm

BTE, 20% Maple tree sap, 308 mOsm

The osmolality of the 20% Maple tree sap was too low to support thecells due to cell swelling.

TABLE 1 Comments on Date Semen was Date Semen was Hen receiving Ratio ofIs this sample Glucose Level in Semen Survival Number Used collected.Sire donating. semen. Semen:Extender. workable? mg/dl and Storage. 4Mar. 31, 2014 Apr. 6, 2011 Squirt Juniper 1 to 2 NO Less than 1% motileon thaw. Most were dead. 5 Apr. 11, 2013 Apr. 9, 2011 Squirt None 1 to 3NO Large sample 40-50 ul. 6 Mar. 25, 2014 Apr. 21, 2011 Squirt None 1 to2 NO Some urate contamination. 0% motility on thaw. 7 X Apr. 23, 2011Squirt None 1 to 2 NO 520 mg/dl Small amount of urate contamination. 0%motility when thawed. On the blood glucose meter it had a 520 mg/dlreading. Impression; There was decreased speed of motility prefreezing.Was the diluent either too thick? 8 Apr. 11, 2013 Apr. 11, 2011 SquirtNone 1 to 2 NO There was a 2 minutes mix time and it went straight tothe tank and was immersed. It was thawed on Apr. 11, 2013 and there was0% motility 9 X Apr. 15, 2011 Squirt None 1 to 4 NO 435 mg/dl Raremotility upon thaw. Less than 1%. 10 Apr. 11, 2013 Apr. 16, 2011 SquirtNone 1 to 4 NO Heavy urate contamination so I diluted with extender sothe semen survives. O % motility on thaw. 11 Mar. 31, 2014 Apr. 16, 2011Squirt None 1 to 1 NO 0% motility on thaw. 12 Apr. 11, 2013 Apr. 24,2011 Squirt 1 to 2 NO 2 cells seen moving. Almost no survivorship 13Mar. 31, 2014 Apr. 25, 2011 Squirt None 1 to 2 NO 0% motility on thaw.14 Mar. 24, 2013 Apr. 26, 2011 Squirt Juniper 1 to 2 NO 0% motility onthaw. 15 Mar. 24, 2013 Apr. 26, 2011 Squirt None 1 to 0 NO BG 5 2%Motile questionable mg/dl, with thawing. retested as 66 mg/dl 16 Mar.31, 2014 Apr. 27, 2011 Squirt None 1 to 2 NO 0% motility on thaw. 17Mar. 24, 2014 Apr. 29, 2011 Jasper None 1 to 2 NO Dies quickly whenviewed as a wet prep w/o freezing. 0% motility upon thawing. 18 Mar. 26,2014 Apr. 25, 2011 Squirt Juniper Unknown NO 1-5% Motility on thaw. 19Mar. 25, 2014 Apr. 30, 2011 Squirt Juniper 1 to 2 NO Tail agglutinationa problem. Survives 3-4 hours at room temperatures. Viewed as a goodsized sample wet prep prior to freezing.; Upon thawing saw 5% or lessmoving and est 1% forward motility. Then put this in Juniper. 20 Mar.25, 2014 May 1, 2011 Squirt None 1 to 3 NO Viewed prior to freezing,tail agglutination is a problem, there is decreased survivorship afterthe extender is frozen in the fridge and used after thawing. Dilutionalso not 1 to 2. Viewed as a trace sample wet prep. Exploded on thaw.Capillary tube found and trace saw 0% motility. 21 Apr. 11, 2013 May 3,2011 Squirt None 1 to 2 NO Good survivorship in diluent w/o freezing.Survived 1:30 PM to 6:50 PM, some survival at room temperature. Whenthawed after freezing there was 0% survival. 22 Apr. 11, 2013 Apr. 6,2013 Squirt None 1 to 3 NO Some urate contamination. 0% motility onthaw. 23 Mar. 30, 2014 May 4, 2011 Squirt Juniper 1 to 2 YES I put thisin Juniper at 1 PM. 25% survivorship with good motility upon thawing. Alot of autoagglutination. Extender frozen prior to use in the freezer.Some survival at room temperature 1 PM to 6:50 PM with little forwardmotility in wet prep. Impressions: Trout #1 seemed better; Extender bestused fresh and not frozen; Cells died faster on this slide and there wasmore agglutination-Stored extender in 5 cc vials and it was colder attime of use. (Important = Cold shock.) 24 Apr. 2, 2014 May 14, 2011Squirt Juniper 2 to 5 MAYBE Heavy urate contamination. On thaw there wasless than 5% motility due to trauma from the explosion of liquidnitrogen. Kevin retrieved this from the can. Used in Juniper the day thetank was filled. 25 Jun. 13, 2013 Apr. 10, 2013 Squirt None 1 to 4 NOMinor urate contamination, 1% good forward motility with a Ice waterthaw. 26 Apr. 11, 2013 Apr. 11, 2013 Squirt None 1 to 3 NO Minor uratecontamination, 0% forward motility on Ice water thaw. 27 Jun. 2, 2013Apr. 11, 2013 Squirt None 1 to 8 NO 0% motility on cold water thaw. Nocryoprotectant used. 28 Jun. 3, 2013 Apr. 12, 2013 Squirt None 1 to 4 NOLost most of the sample. Unable to evaluate. No survival seen in onesdiluted with water. Cold water thaw. 29 Mar. 31, 2014 May 7, 2011 SquirtJuniper 1 to 3 NO Less tail agglutination with this formula. More rapidcell death though after only 20 minutes. Maybe 30% survival, 5% forwardmotility at 1 hour. None alive on wet prep at 6:50 PM, (collected 1 PM),impression, sperm dies fast in this extender. Less than 1% motile onthaw and put in Juniper Mar. 31, 2014. 30 Apr. 14, 2013 Apr. 4, 2013Squirt 1 to 2 NO No motility seen on fresh wet prep. 7 very mobile cellsseen, 1 cell was moving fast and then slowed down and stopped. I usedTrout #2 plus ¼ tsp Sorbitol and ⅛ tsp Arabogalactin. 31 Mar. 30, 2014May 30, 2013 Squirt Juniper 1 to 4 MAYBE Trace sample after explodingsaw some motility 32 X 2014 May 31, 2013 Squirt Juniper 16% NO 8 mm ofSemen placed in room temperature Turkey Extender went to the fridge for30 minutes acclimation time, Added 3 units DMA for a final volume of 50mm. Quickly placed over liquid nitrogen in under 60 seconds. 33 X 2014Jun. 1, 2013 Squirt Juniper 44% NO 22 mm of Semen was placed in TurkeyExtender at room temperature and placed in the fridge and acclimated for30 minutes. 3 units/ul of DMA was added. I was placed over the liquidnitrogen in under 60 seconds. It was hung over the vapors for 10 minutesand then flash frozen. 34 Mar. 29, 2014 Jun. 2, 2013 Squirt Juniper 48%NO 24 mm of Semen was placed in Turkey Extender at room temperature to avolume of 47 mm and then acclimated in the fridge for 30 minutes. 3 ulof DMA was added quickly and then the sample was hung over the vapor (inless than 60 seconds) for 10 minutes and then immersed in liquidnitrogen. 35 Apr. 3, 2014 May 28, 2013 Squirt Juniper 20% NO 8-10 mm ofSemen was collected and Turkey Extender was added that was at fridgetemperatures. The final volume with DMA was 50 ul/mm. 35 mm of Extenderand 5 ul of DMA was used to make it 10% DMA. “No motility” (probably toocold) seen on the smear on the fresh wet prep. When thawed on ice water5-10% were seen moving. Only 1% with good not great forward motility.There was progressive loss of motility over minutes. (45 minutes). Thisis the likely sample that went into Juniper on 4/3. 36 X 2014 May 29,2013 Squirt Juniper 8% 4 mm of Semen was collected and Turkey Extenderthat was refrigerator temperature was added to a volume of 47 mm. Thiswas left in the fridge at 40 F. from 7 AM to 6:30 PM, 3 ul of DMA wasadded quickly and in less than a minute it was hung over the vapors for10 minutes and then immersed. 50% great motility off trace sample seenbefore adding the DMA. 37 Mar. 26, 2014 Jun. 3, 2013 Squirt None 8% 4 mmof Semen was collected and Turkey Extender was added to a volume of 45mm. It was acclimated for 30 minutes and then 5 ul (10%) DMA was added.It was Flash Frozen. 38 Mar. 26, 2014 Jun. 3, 2013 Squirt None 8% NO 2mm of Semen was collected and Turkey Extender 23 mm was added and it wasacclimated in the fridge for 30 minutes. (10%) DMA was added quickly andit was Flash Frozen in under a minute. 39 Mar. 26, 2014 Jun. 4, 2013Squirt None 8% NO 4 mm of Semen was collected and 45 mm of TurkeyExtender was added and it was acclimated in the fridge for 30 minutes. 5ul of DMA was added (10%) and it was Flash Frozen after mixing in undera minute. 40 Mar. 26, 2014 Jun. 5, 2013 Squirt None 4% NO 1 mm of Semenwas collected and 8 mm of Turkey Extender was added and it wasacclimated in the fridge for 30 minutes. 2 ul of DMA was added and itwas flash frozen after mixing in under a minute. 41 2014 2013 4 SquirtJuniper YES 4 TUBES THAT WERE SAMPLES PRESERVED IN 2013 FLOATING THATWERE PUT IN IN THE COMMERCIAL PLASTIC LIQUID TUBES WITH BEADS TONITOGEN. CAP THE ENDS, SEMEN WAS IN CAPPILLARY TUBES, WERE FOUNDFLOATING IN THE LIQUID NITROGEN ON TOP. BUT LOST THEIR LABELS. SOME OFTHESE SAMPLES CONTAINED 25% SURVIVAL OF SEMEN SAMPLES AND WERE ACTIVELYMOVING FORWARD. THESE WERE PUT INTO JUNIPER. ONE OF THE RED AND FOUR OFTHE GREEN SAMPLES APPEAR TO BE WHAT I USED FOR AI IN JUNIPER. I DO NOTKNOW WHICH TUBE WAS WHICH BUT I DO KNOW THAT I LOST 3 RED SAMPLES ONTHAW DUE TO EXPLODING. THE REMAINING SAMPLES DID NOT LEAK IN THE PRIMARYCONTAINER. MOST OF THE GREEN SAMPLES WERE NOT LOST. THE ENTIRE TANK WASEMPTIED OF ALL OF ITS SAMPLES IN 2014. STARTING OVER. 16 SAMPLES FOR2013 PUT IN THE TANK. 42 May 2, 2014 Squirt 3 mm No urates. Lowcellularity. Semen to 30 units of Turkey extender, plus 1.5 units DMA 43May 1, 2014 Squirt 6 mm Some urate Semen, contamination. Low diluted tocellularity. 30 mm volume, added 1.5 units DMA 44 Mar. 1, 2016 May 3,2014 Squirt NO No urates. Low cellularity. Mar. 1, 2016 Thawed in an icewater bath. Low cellularity due to males age. Less than 10% motile. Cannot do a Live/Dead stain as cellularity is too low. 45 Mar. 29, 2015 May4, 2014 Squirt 2 mm of NO No urates. Low cellularity. Semen Goshawksemen requires plus 18 mm sucrose and not fructose of Turkey to survivefreezing!!! This extender to is why these cells survived a total in theTrout Extender #2 volume of and not the Turkey 20 mm. Extender that hasthe Fructose! You must use Beltsville Turkey Extender minus theFructose, with sucrose added back in to .5% (½%) 46 May 4, 2014 Squirt 6mm of Low number of urates. Semen, Low cellularity. plus 19 mm of TurkeyExtender, plus 2 ul of DMA. 47 May 5, 2014 Squirt 1-40 mm 1 side of thesplit sample sample had more urates than the contaminated other, semenin only one with spot on the tube, so urates split separated into ½ toput into 2-20 most urates in 1 tube. Low mm cellularity. samples, plus28 mm Turkey extender, plus 3 ul of DMA (6%). 48 May 6, 2014 Squirt 6 mmof No urates. Low cellularity. Semen, plus 14 mm of Turkey extender to atotal volume of 20 mm. Plus 1.5 ul of DMA, (7%) 49 May 6, 2014 Squirt10-16 mm Many urates. Small of Semen amount of semen in one plus 47 mmspot. Final volume 50 mm. of Turkey Low cellularity. extender, plus 3 ulof DMA. 50 May 7, 2014 Squirt 6 mm of No urates. Low cellularity. semenplus 19 mm of Turkey extender plus 2 ul of DMA. 51 May 7, 2014 Squirt 6mm of No urates. Low cellularity. Semen Lots of blast cells seen. plus19 mm Goshawk semen requires of Turkey sucrose and not fructose extenderto survive freezing!!! This plus 1.25 ul is why these cells survived ofDMA in the Trout Extender #2 (5%). and not the Turkey Extender that hasthe Fructose! You must use Beltsville Turkey Extender minus theFructose, with sucrose added back in to .5% (½%) 52 Mar. 29, 2015 May 9,2014 Squirt None 5 plus mm NO Watery urates seen in 8 of of Semen 13 mmtotal initial semen and about volume so had about 5 8 mm of mm of semenpresent. Urates, Low cellularity. Goshawk plus 27 mm semen requiressucrose of Turkey and not fructose to extender to survive!! These cellscan a total not use fructose and this is volume of why these cellssurvive in 40 mm plus the Trout #2 extender! 2.7 ul of DMA (6%) 53 May10, 2014 Squirt 16 mm of No urates and cellularity is Semen low butgoing up. plus 22 mm of Turkey extender for a final volume of 38 mm,plus 2.23 ul of DMA, (6%). 54 May 22, 2014 Squirt 5 mm of No urates andcellularity is Semen low but going up. plus 15 mm of Turkey extender toa final volume of 2 mm, plus 1 ul of DMA (5%). 55 May 12, 2014 Squirt 14mm of A few urates, but not a lot. Semen Low cellularity but plus 23 mmincreasing. Turkey extender to a total of 47 mm. Plus 2.5 ul of DMA(5%). 56 May 14, 2014 May 13, 2014 Squirt None 4 mm of No Unknown, Nourates. Cellularity is Semen meter low but climbing. plus 20 mm couldnot Storage straw did not leak of Turkey read this and the cellsurvivorship extender number. was feeble. plus 5% Maple Syrup, plus 1 ulDMA (5%). 57 Apr. 4, 2015 Mar. 22, 2015 Odin None 8 mm of No No semensurvived Semen freezing. The fructose in plus 11 ul the sample is the ofBeltsville suspected problem Turkey because it slows the Extender speedof the spermatozoa (unaltered) down by half in fresh Plus 1 ul ofsamples extended with DMA. Total this TE. The volume cryoprotectantneeds to 20 ul. also be looked at. All 2015 samples placed in coldEppendorf tubes that were already in the fridge. Temperature shock mightbe present. Might be too cold next to refrigerator coils. Goshawk semenrequires sucrose and not fructose to survive freezing!!! This is whythese cells survived in the Trout Extender #2 and not the TurkeyExtender that has the Fructose! You must use Beltsville Turkey Extenderminus the Fructose, with sucrose added back in to .5% (½%) 58 Apr. 18,2015 Mar. 23, 2015 Odin None 30 ul of NO All 2015 samples placed inSemen cold Eppendorf tubes that plus 30 ul were already in the fridge.of Turkey Temperature shock might extender be present. Might be too pluscold next to refrigerator Inositol. coils. Lost sample across Plus 2.6ul the garage as it exploded. of DMA, 62.6 ul total volume. 59 Apr. 18,2015 Mar. 23, 2015 Odin None 30 ul of NO A sample that was still leftSemen in the tube before freezing plus 30 ul had 25% survival based ofTurkey on a live/dead stain. All extender 2015 samples placed in pluscold Eppendorf tubes that Inositol. were already in the fridge. Plus 2.6ul Temperature shock might of DMA. be present. Might be too 62.6 ultotal cold next to refrigerator volume. So coils. Less than 1% this is4% Motile with 55 F. water bath DMA. and hand warming. 60 Apr. 18, 2015Mar. 25, 2015 Odin None 12 ul of NO All 2015 samples placed in Semencold Eppendorf tubes that plus 24 ul were already in the fridge. ofTurkey Temperature shock might Extender be present. Might be too pluscold next to refrigerator Inositol. coils. No survival on Plus 2 ul ofthawing. Thawed in a cool DMA. 38 ul water bath at 55 F. and total handwarming. volume. So this is 5% DMA 61 Apr. 18, 2015 Mar. 25, 2015 OdinNone 10 ul of NO All 2015 samples placed in semen plus cold Eppendorftubes that 18 ul of were already in the fridge. Turkey Temperature shockmight extender be present. Might be too plus cold next to refrigeratorInositol, coils. Thawed in cool plus 2 ul of water bath at 55 F. and DMATo a hand warming. No cell total survival. volume of 30 ul. 6.6% DMA 62Apr. 15, 2015 Mar. 27, 2015 Odin None 15 ul of NO All 2015 samplesplaced in Semen cold Eppendorf tubes that plus 30 ul were already in thefridge. of Beltsville Temperature shock might Turkey be present. Mightbe too Extender cold next to refrigerator plus coils. Thawed in coolInositol, water bath at 55 F. and plus 2 ul of then put on a warmingDMA, or plate. Less than 1% 4.25% survival. DMA 63 Apr. 15, 2015 Mar.27, 2015 Odin None 20 ul of NO All 2015 samples placed in Semen coldEppendorf tubes that plus 37 ul were already in the fridge. ofBeltsville Temperature shock might Turkey be present. Might be tooExtender cold next to refrigerator plus coils. Thawed in a coolInositol, water bath at 55 F. No plus 3 ul of cells survived. DMA, 5%DMA 64 Apr. 15, 2015 Mar. 28, 2015 Odin None 16 ul of NO All 2015samples placed in Semen cold Eppendorf tubes that plus 29 ul werealready in the fridge. of Beltsville Temperature shock might Turkey bepresent. Might be too Extender cold next to refrigerator with coils.Thawed in a cool Inositol, water bath at 55 F. No plus 3 ul of cellssurvived. DMA, 6.25% DMA 65 Apr. 15, 2015 Mar. 28, 2015 Odin None 14 ulof NO All 2015 samples placed in Semen in cold Eppendorf tubes that 16ul of were already in the fridge. Beltsville Temperature shock mightTurkey be present. Might be too Extender cold next to refrigerator pluscoils. Thawed in a cool Inositol, water bath at 55 F. and plus 2 ul ofthen put on a warming DMA, plate. No cells survived. 6.25% DMA 66 Apr.15, 2015 Mar. 28, 2015 Odin None 13 ul of NO All 2015 samples placed inSemen in cold Eppendorf tubes that 20 ul of were already in the fridge.Beltsville Temperature shock might Turkey be present. Might be tooExtender cold next to refrigerator plus coils. Inositol, plus 2 ul ofDMA, 5.7% DMA. 67 Mar. 30, 2015 Mar. 29, 2015 Odin None 16 ul of MAYBE 5times This sample was flash Semen higher frozen with no acclimation.plus 24 ul than BTE When I thawed it, it took of BTE, no normally offliving just like the fructose, is so this sample did that was plus Mapleis viewed at room tree sap, hyperosmolar. temperature. There was noother about 11% motility and cryoprotectant less than half of these weremoving forward well on thaw. A sample that was left on a slide at roomtemperature had the mature spermatids stop moving within 5 minutes butthe immature spermatids did well and kept on moving. The speed ofmovement was much better and this is clearly an improvement over the BTEwith fructose. I can assume that goshawk semen needs sucrose to survive.The sap is 100 milliosmoles and was added to the dry ingredients of theBTE, no fructose. This added in sucrose at 5 times the percentage neededand made it hyperosmolar. The caused the mature cells to die. Theimmature spermatids, with immature cell walls, could equalize theosmotic pressure. Maple sap is .5-2.6% sucrose. This is first run sap soit is higher in sucrose than last run. Different maple tree species havedifferent percentages of sucrose. 68 Apr. 15, 2015 Mar. 29, 2015 OdinNone ? NO 6% DMA, No survival 69 Mar. 31, 2015 Mar. 29, 2015 Odin None19 ul of MAYBE, Sucrose Most of the sample was Semen MORE likely 5 loston thaw. The trace in plus 38 ul SUCCESS times the sample has 66% of BTEplus THAN I what is motility based on visual Maple tree HAD needed,estimates. 25 out of 43 sap, first HOPED so this alive on a swim countwith run, tree FOR. sample is the counter. On a live number 3.hyperosmolar. dead stain there was Live No other 58/42 Dead!cryoprotectant 70 Apr. 15, 2015 Apr. 3, 2015 Odin None 25 ul of YES, butSucrose This tube is completely Semen, can do likely 5 full. 5-10%motility on thaw plus BTE, better. times with normal motility. nofructose, what is Thawed in 55 F. water bath plus 50 ul needed, and thenplaced on a of Maple so this warm plate. tree sap, sample is No otherhyperosmolar. cryoprotectant. 71 Apr. 15, 2015 Apr. 3, 2015 Odin None 16ul of YES Sucrose Thawed in 55 F. water bath Semen likely 5 and thenplaced on a plus BTE, times warming plate. 10% no fructose, what ismotility at thaw and then plus 28 ul needed, the cells slow theirmotility of Maple so this to 5% estimated visually tree sap, sample isover 5 minutes. No other hyperosmolar. cryoprotectant. 72 Apr. 15, 2015Apr. 4, 2015 Odin None 13 ul of YES Sucrose Thawed in a 55 F. waterSemen likely 5 bath and then a warming plus BTE, times plate. 20% nearnormal no fructose, what is motility and motility plus 26 ul needed,estimated visually. of Maple so this Thawed fast! Ventilated Tree sap,sample is soda straw is key. no other hyperosmolar. cryoprotectant wasused. 73 Apr. 15, 2015 Apr. 12, 2015 Odin None 20 ul of NO Sucrose Atroom temperature, cells Semen level slow rapidly on a wet prep, plus 40ul reads at probably still too of BIRCH 479 mg/dl hyperosmolar. Nomotility tree sap on a on thaw with 55 F. water Accucheck bath and thena warming glucose plate. meter. 74 Apr. 14, 2015 Apr. 12, 2015 Odin NONE42 ul of MAYBE Apr. 14 2015 Sample #1 Semen, Orange straw; Thawed inplus BTE, cold water and then no fructose, placed on a warming plusplate. 5-10% motility of Sucrose, normal looking sperm. The Added inrest are moving a little but 75 ul of not swimming forward. BTE, minusfructose, plus sucrose ½% plus MA (methyacetamide) 75 Apr. 13, 2015 Odin45 ul of Semen split between 3 straws, BTE no fructose, plus ½% sucrose,plus 84 ul of BTE (same as above) with MA (Methyacetamide) 76 Feb. 28,2016 Apr. 14, 2015 Odin None 7 ul of Yes Sample was prepped at Semenroom temperature with with 26 ul all items starting at 70 F. of BTE, noFeb. 28, 2016 Teal Blue fructose, Straw, Not ventilated, plus ½% 80% ofcells vibrating, sucrose. 10% moving actively, Plus 2 ul Thawed in a 41F. water of DMA bath. Live 65/45 Dead cryoprotectant. Stain. Finalvolume of 35 ul. 77 Feb. 28, 2016, Apr. 14, 2015 Odin None 55 ul of Thisis All materials start at room Mar. 1, 2016 Semen one of the temperature(70 F.) and plus 110 ul first then go to fridge to chill to of BTE, nosamples 41 F. Cold packs were fructose, of NO used to carry to the plus½% FRUCTOSE garage. sucrose, plus Feb. 28, 2016 Thawed in 41 F. chilledin DMA to water bath to a warming the fridge test if it is plate withLive 39/61 for 10 the Dead. minutes, fructose Mar. 1, 2016 Thawed in aplus 8 ul of or DMA 41 F. water bath to a DMA that is warming plate with10% causing swimming normally and the 50% vibrating in place. samples tofail. Feb. 28, 2016 Yes, No movement- green tube, Thawed 41 F. waterbath. Little cell deformity with a Live 39/61 Dead stain. Samplequiescent. Mar. 1, 2016 Yes, 10% are swimming normally, 50% arevibrating, Thawed in a 41 F. water bath. Live 32/68 Dead stain. 78 Apr.18, 2015, Apr. 15, 2015 Odin None 38 ul of Maybe, Acclimated at 41 F. inApr. 18, 2015 Semen, because fridge with sap separate plus 75 ul the sapfrom semen until placed in of Birch preserves capillary tubes for Sap,BTE, the freezing. Acclimated for 10 no fructose, semen minutes. firstrun.. when Apr. 18, 2015 Thawed in a 55 frozen. F. water bath. Sample 1But the showed 5% spermatozoa amount of moving forward normally sap witha live/dead stain of needs to 32/68. be May 18, 2015 Sample 2 hadreduced. about 5% moving forward Similar normally with a live/deadresponse stain of 34/66. to Maple tree sap. The cells survive the freezebut stop moving due to hyperosmolality. 79 Apr. 18, 2015, Apr. 17, 2015Odin None 28 ul of Maybe, This was first run Birch Apr. 18, 2015 Semen,because tree sap from Alaska. It plus 28 ul the sap was acclimated afterBTE, no preserves mixed by only placing it fructose, the between gelpacks that plus ½% semen were at 41 F. from the sucrose, when fridge andthen it was flash plus 56 ul frozen, frozen. Two straws were Birch Treebut the made from this sample. sap in BTE, amount of One was a yellow,and the no fructose, sap other green, ventilated final needs to sodastraws. One tube volume of be had 2-5% forward moving 112 ul withreduced. sperm on visual estimate 1:3 semen Similar with a live/deadstain of to Birch response 21/79. The second tube tree sap to Maple had0% forwardly moving extender. tree sap. and no live dead stain was Thecells done. Thawed in a 55 F. survive cool water bath. the freeze butlose motility due to hyperosmolality. 80 Apr. 18, 2015, Apr. 17, 2015Odin None 50 ul of Maybe, Thawed in a cool water Apr. 18, 2015 Semen hadMaybe bath of 55 F. 50 ul of Apr. 18, 2015 First sample 2-3% BTE, nonormal motility, with a fructose, live/dead stain of 57/43.; plus 1/%Apr. 18, 2015The second sucrose sample had 5-10% added moving forwardnormally together in and a live dead stain of 1 tube. 42/58. Later 100ul of BTE, no fructose, plus Maple sap was added for a final volume of200 ul. Making the semen ¼ of the total mix. 81 Apr. 18, 2015, Apr. 18,2015 Odin None 22 ul of YES, Thawed in a cool water Apr. 18, 2015 Semenhad YES bath of 55 F. 22 ul of Apr. 18, 2015 First sample BTE, no had25-30% normal fructose, motility with a live dead plus ½% stain of50/50.; sucrose Apr. 18, 2015 The second added to it sample had about55% placed in 1 normal forward motility tube. Later with a live/deadstain of 44 ul of 57/43. 100 cells were BTE, no counted in each group.fructose, plus Maple tree sap was added for a final volume of 88 ul. 82May 10, 2015, Apr. 20, 2015 Odin None 65 ul of Yes, Acclimated at 41 F.in Feb. 24, 2016, Semen, Maybe, fridge with sap separate Green plus 65ul No, No from semen until placed in straw- BTE minus capillary tubesfor Holder 5, fructose, freezing. Acclimated for 10 Mar. 2, 2016 plus ½%minutes. Then flash Green sucrose. frozen. Ice water thaw, straw- Later130 10-15% good motility, Live Holder 6, ul of BTE Dead Stain 24 live/76Mar. 2, 2016 minus dead. Green fructose Feb. 24, 2016 Thawed in a 41straw plus Birch F. ice water bath, 2-3% Holder 6. tree sap movingforward well, pH first run. 7.0-7.2 with a Live22/78 Dead Stain. Mar. 2,2016 Thawed in a 41 F. Ice water bath, Green Straw Holder 6, Rare motilesperm with a Live 10/90 Dead Stain. Cells are very distorted. Mar. 2,2016 Thawed in an ice water bath at 41 F. No motility and the cells arevery deformed. Live 7/93 Dead. 83 May 3, 2015, Apr. 20, 2015 Odin None60 ul of No, No Acclimated at 41 F. in Mar. 6, 2016, Semen, Mar. 6, 2016fridge with sap separate Mar. 6, 2016 plus 60 ul Yellow from semen untilplaced in of BTE − straw, capillary tubes for fructose, + water gotfreezing. Acclimated for 10 ½% into minutes. Then flash sucrose. sample.frozen. Later 60 ul YES May 3, 2016 Thawed in Ice of BTE plus Mar. 6,2016 water, 2% forward Maple tree Yellow motility, Live dead stain, sap,first straw 39 live/61 dead. run Tree Mar. 6, 2016 Thawed in ice #3.added water at 41 F. 1% Motile for a final and 1% moving in place.volume of Live 33/67 Dead stain. A 180 ul. lot of agglutination. It wasexposed to water on thaw because it lost the caulk on the end of thetube. Mar. 6, 2016 Thawed in a ice water bath. 15-20% are motile.Agglutination is present. Live 49/51 Dead. 84 May 3, 2015, Apr. 21, 2015Odin None 50 ul of YES, Acclimated at 44 F. in Mar. 1, 2016, Semen hadYES, fridge with sap separate Mar. 6, 2016 50 ul of YES from semen untilplaced in BTE, no capillary tubes for fructose, freezing. Acclimated 10plus 1/% minutes. Then flash sucrose frozen. added May 3, 2015 Thawed inIce together in water bath, 55%-60% 1 tube. forward motility, Live deadLater 100 stain 39/61. No pH done. ul of BTE, Mar. 1, 2016 Thawed in anno fructose, ice water bath at 41 F., plus Maple Over half are movingsap was forward with fast motility, added for a pH of 7, with a Live64/36 final Dead stain. volume of Mar. 6, 2016 Thawed in an 200 ul. icewater bath at 41 F. Making the Over 60% are motile with semen ¼ littledeformity. They have of the total fast motility with a Live mix. 73/27Dead stain. 85 Mar. 2, 2016, Apr. 21, 2015 Odin None 50 ul of Maybe -Acclimated at 44 F. in Mar. 2, 2016 Semen had Quiescent, fridge with sapseparate 50 ul of Maybe from semen until placed in BTE, no Quiescentcapillary tubes for fructose, freezing. Acclimated 10 plus 1/% minutes.Then flash sucrose frozen. added Mar. 2, 2016 Thawed in an together inice water bath and put on 1 tube. a warming plate. Had 5% Later 50 ulforward motility and a Live of BTE, no 36/64 Dead stain. fructose, Mar.2, 2016 Yellow straw, plus Maple Thawed in an ice water sap was bath at41 F. Less than 1% added for a are motile with a Live final 40/60 Deadstain. volume of 150 ul. Making the semen ⅓ of the total mix. 86 May 3,2015, Apr. 21, 2015 Odin None 10 ul of No, No acclimation in the May 3,2015 semen plus Maybe fridge. Flash frozen. 10 ul of May 3, 2015 Firstsample BTE − Fruc, + thawed in ice water, No ½% motility, pH of 7 on pHsucrose, paper, May 3, 2015 Second PLUS sample thawed in ice YOLK,water, 2-5% motility, pH 7 Later 12 ul on paper. Live dead stain of BTE− 48 live/52 dead. Fruc, Plus Maple sap, first run tree 3. 87 Mar. 7,2016 Apr. 22, 2015 Odin None 16 ul of Maybe - Acclimated in the fridgein Semen, Quiescent. separate tubes at 43 F. plus 16 ul The Mixed,packaged, and BTE − Fruc, + cells are then flash frozen. ½% not Mar. 7,2016 Pink soda straw sucrose, distorted 1% Motile/Cells not PLUS butlikely distorted. Live 19/81 Dead YOLK, quiescent stain. Later 16 ul andnot BTE − Fruc, motile. plus Maple sap, plus 10% Yolk. Final volume 48ul. 88 May 3, 2015, Apr. 22, 2015 Odin None 53 ul of Yes, No Acclimatedin the fridge in Mar. 2, 2016 Semen, Thawing separate tubes at 43 F.Pink straw plus 53 ul too warm. Mixed, packaged, and Holder #6, of BTE −No Cold then flash frozen. Mar. 6, 2016 fructose, + water May 3, 2015Thawed in Ice Pink straw ½% thaw is no water, 10% moving Holder #6.sucrose, better. forward, pH of 7. Live PLUS dead stain, Live 38/62YOLK, Dead. Later 53 ul Mar. 2, 2016 Thawed in a of BTE − 55 F. waterbath, Less than fructose, + 1% motile, many cells Maple sap, deformedwith a Live plus 10% 16/84 Dead stain. YOLK Mar. 6, 2016 Thawed in anice water bath, 0% motile, Cells not deformed with a Live 13/87 Deadstain. 89 May 10, 2015, Apr. 23, 2015 Odin None 45 ul of No, NoAcclimated in the fridge in Mar. 2, 2016 Semen with separate tubes at 43F. 55 ul of Mixed, packaged, and BTE − then flash frozen. fructose, +May 10, 2015 A trace sample ½% of this tube survived well sucrose atroom temperature. pH and YOLK of 7, Ice water bath thaw, added in atLess than 1% motile, Live 10%, Later dead stain could not be 35 ul ofdone. Could not read the BTE + Live/Dead stain due to the Birch Treeyolk being present in the Sap so that sample. it becomes Mar. 2, 2016Orange tube, 26% Birch Thawed in an ice water Tree Sap. bath, Less than1% motile with a Live 33/67 Dead stain. Many cells are deformed. 90 May3, 2015, Apr. 23, 2015 Odin None 45 ul of Maybe, Acclimated in thefridge in Mar. 1, 2016, Semen with Maybe separate tubes at 43 F. Mar. 2,2016 55 ul of appears Mixed, packaged, and Holder 6 BTE − Quiescent.then flash frozen. Green fructose, + Maybe May 3, 2015 Thawed in a Straw½% appears 55 F. water bath. Less than sucrose Quiescent. 1% motile orno motility. and YOLK Mar. 11, 2016 Thawed in a ice added in at waterbath, No motility but 10%, Later the cells are not deformed 32 ul ofwith a Live 34/66 Dead BTE + stain = Quiescence. Birch Tree Mar. 2, 2016Thawed in a ice Sap so that water bath. No motility but it becomes thecells are not deformed 19.7% with a Live 31/69 Dead Birch Tree stain =Quiescence. Sap. 91 Feb. 24, 2016 Apr. 24, 2015 Odin None 30 ul ofMaybe, Mixed at room Green soda Semen, Maybe temperature and then strawfrom with 60 ul appears placed between gel packs Holder #4, of BTE −Quiescent. at 43 F. Then flash frozen. Feb. 24, 2016 fructose, + Feb. 242016 Green soda Deep blue ½% straw from Holder #4 soda straw sucrose.Thawed in an ice water from Holder Later 30 ul bath at 41 F. Cells not#4. of BTE − deformed and 2-3% fructose, motile with a Live 23/77 plus½% Dead stain. sucrose Feb. 24 2016 Deep blue plus 18% straw from Holder#4, MA (diluted Thawed in an ice water to a total bath at 41 F., Cellsnot percentage deformed 80% and 1% of 4.5% motile. But this tube had MA)10-20% deformed cells overall with a Live 35/65 Dead stain. 92 Feb. 28,2016 Apr. 25, 2015 Odin None 15 ul of Maybe Mixed at room Semen appearstemperature and then plus 15 ul Quiescent placed between gel packs ofBTE − with 10% at 43 F. The flash frozen. fructose, + movement. Feb. 28,2016 Thawed in an ½% ice water bath at 41 F., sucrose, ventilated sodastraw with plus 15 ul 10% moving forward and a of BTE − Live 35/65 Deadstain. fructose, + Little cell distortion. ½% sucrose plus 18% MA (finalof 6% MA). 93 Feb. 24 2016, Apr. 24, 2015 Odin None 45 ul of Yes, Yes,Chilled 10 minutes at 45 F. Green Semen, Yes Mixed packaged and thenstraw with 45 ul flash frozen. Holder #4, of BTE − Feb. 24 2016 Greenstraw Feb. 24, 2016 fructose, + Holder #4 Thawed in an Green ½% icewater bath at 41 F., cells straw sucrose, not deformed with 2-3% Holder#4, Later 45 ul motile. Quiescent but alive Feb. 24, 2016 of BTE, − withLive 55/45 Dead Green fructose + stain. straw ½% Feb. 24 2016 Green sodaHolder #4. sucrose straw Holder #4 Thawed in plus 18% an ice water bathat 41 F., MA = 6% cells not deformed, 25- MA final 30% motile and showedconcentration slowing motility over 5 minutes with a Live 57/43 Deadstain. Feb. 24 2016 Green soda straw from Holder #4. Thawed in an icewater bath at 41 F. Cells were not deformed and had 5% motile withslowing of motility over 5 minutes. The live/dead stain was hard to readbut had Live18/82 Dead. 94 May 10, 2015, Apr. 25, 2015 Odin None 20 ulof No Chilled for 10 minutes and May 10, 2015 Semen then added DMA. Iceplus 55 ul water bath thaw. Almost of BTE − no motility, No live deadfructose, + stain done. Assessment; ½% DMA is not working. sucrose.Later 4.9 ul of DMA added. 95 May 3, 2015, Apr. 26, 2015 Odin None 22 ulof No, I AM STARTING THE Mar. 6, 2016 Semen Maybe PURDY FORMULAS Orangeplus 66 ul THAT ARE BTE WITH straw of BTE + DIFFERENT 10% MaplePERCENTAGES OF Sap MAPLE TREE SAP IN PURDY THE DILUTION. THESE FORMULA.HAVE FRUCTOSE AND SUCROSE IN THEM. SEE SHEET ON THESE FORMULAS. May 3,2016 Ice water bath thaw, 2% moving forward, pH of 7 on paper. Mar. 6,2016 Thawed in an ice water bath at 41 F. Orange straw with 2% movingforward and 2% moving in place with a Live 14/86 Dead stain. 96 May 10,2015 Apr. 27, 2015 Odin None 18 ul of No May 10, 2016 Ice water bathSemen with thaw, 1-3% of these 54 ul of moving forward. Live dead Purdy10% stain 31 live/69 dead. Maple tree Comments; Fructose, No sap. 1:3acclimation, and high pH dilutions. might be a problem. 97 Mar. 6, 2016Apr. 27, 2015 Odin None 12 ul of YES! Mar. 6, 2016 No acclimation Pinksoda Semen and was thawed in an ice straw plus 36 ul water bath. Greaterthan Holder #6 of Purdy 10 50% moving, with 20% % Maple moving normally.There Tree sap. was a Live 52/48 Dead stain. 98 Purdy Purdy Purdyformulas begin with Formulas formulas #95. begin with #95 99 Feb. 24,2016 Apr. 27, 2015 Odin None 50 ul of Maybe Processed at room Holder #5Semen with sap temperature, placed Pink soda 100 ul of appears between43 F. gel packs, straw, Purdy 10% to and then flash frozen. Feb. 24,2016 Maple tree decrease Feb. 24, 2016 Pink straw Holder #4 sap.movement = Holder #5 Thawed in an Pink soda quiescence. ice water bathat 41 F., No straw, Maybe = movement, no pH done, Feb. 24, 2016 Same,Cells very distorted. Had a Holder #4 Maybe = Live27/73 Dead stain. Pinksoda Same. Feb. 24, 2016 Pink soda straw straw. Holder #4 Thawed in anice water bath at 41 F. Almost no movement and cells very distorted. Hada Live 29/71 Dead stain. Feb. 24, 2016 Pink soda straw Holder #4 Thawedin ice water bath at 41 F. Less than 1% moving forward and cells weredistorted. Live 36/64 Dead. 100 May 16, 2015, Apr. 28, 2015 Odin None 70ul of Maybe Processed at room Mar. 6, 2016, Semen Quiescent,temperature, placed Mar. 7, 2016 plus 210 ul Maybe between 43 F. gelpacks, Green of Purdy Quiescent, and then flash frozen. straw from BTE +20% No, No May 16, 2015 Thawed in ice Holder #6, Maple tree water, 1-5%moving well Mar. 13, 2016 sap 308 but motility slows quickly. GreenmOsm. Live dead stain shows 39 straw from live/61 dead, 30 live/70Holder #6. dead. Hot plate on microscope appears to speed loss ofmotility. Mar. 6, 2016 Thawed in an ice water bath at 41 F. 10- 15%motility swimming forward. Many of the cells are distorted due to thelow osmolality. There is a Live 17/83 Dead stain. Mar. 7, 2016 Greenstraw. Thawed in an ice water bath. Had 1-2% moving. The cells are verydistorted and it had a Live 20/80 Dead stain. Mar. 13, 2016 Thawed in anice water bath and the cells are very distorted. Less than 1% motilityand a Live 9/91 Dead stain. 101 May 3, 2015, Apr. 29, 2015 Odin None 20ul of Unknown Mar. 2, 2016 Thawed in an Mar. 2, 2016 Semen as ice waterbath at 41 F. Yellow plus 60 ul sample Yellow straw. Cells very straw,Purdy 10%, from deformed. Live 3/97 Dead Holder #6 Maple sap holder #5stain. was lost from straw., Maybe 102 May 10, 2015, Apr. 30, 2015 OdinNone 52 ul of Maybe, Processed at room Mar. 2, 2016 Semen Maybe,temperature, placed Orange plus 104 ul Maybe- between 43 F. gel packs,straw from of Purdy Many for 2 minutes and then Holder #6, 5% Mapleswollen flash frozen. Mar. 6, 2016 Sap and May 10, 2015 Thawed in 70 F.Orange distorted water bath, Live dead straw from cells. stain; 28live/72 dead, Holder #6. Mar. 2, 2016 Orange soda straw. Thawed in anice water bath at 41 F. Had 10% forward motility with many deformedcells. The speed of motility increased with warming. It had a Live 33/67Dead stain. Mar. 6, 2016 Thawed in an ice water bath at 41 F. It had10-15% forward motility and many distorted and swollen cells. Live 21/79Dead. Motility speed increased with warming. 103 May 3, 2015, Apr. 30,2015 Odin None 65 ul of NO, No acclimation in the May 10, 2016, Semen,maybe, fridge. Flash frozen. Mar. 21, 2016 plus Purdy Yes = Thawed May3, 2015 in Ice Green 10% Maple Quiescent, water, pH 7.5, 0% motility,straw Sap, plus Yes = no live dead stain done, Holder #6, 5% (13 ul)Quiescent Sample thawed May 10, 2015 Mar. 13, 2016 of DMA, No Ice waterbath, pH 7, 2-3% Green acclimation motility, Live Dead Stain straw Live23/Dead 77. Holder #6 Mar. 2, 2016 Thawed in an ice water bath at 41 F.Saw 2% motile Almost no gross cell deformity with a Live 52/48 Deadstain. The longer it warmed on the plate the higher the motility up to4%. Mar. 13, 2016 Thawed in an ice water bath at 41 F. Less than 1%motility. Little cell distortion and a Live 20/80 Dead stain. 104 May 3,2015 May 1, 2015 Odin None 8 ul of No May 3, 2015 Cells do not do Semenwell with this at room plus 8 ul temperature. Thawed in Purdy 10% icewater, less than 2% Maple tree motile, No live/dead stain sap plus done.Arabogalactin, (No MA), Plus Purdy 10% Maple Sap plus Arabogalactin plus12% MA 105 Apr. 4, 2016, Mar. 25, 2016 Odin None 55 ul of Maybe; Apr. 4,2016 Thawed in a ice Apr. 8, 2016, Semen; Mistake water bath. Nomotility and Apr. 10, 2016 plus 55 ul made has a Live 54/46 Dead of BTEadjusting stain. with/out pH up in Apr. 8, 2016 Thawed in an fructose2nd ice water bath at 41 F. and plus ½% media then palmed to warm. NoSucrose; used - motility with a Live 4/96 plus 55 ul using Dead stain.of BTE w/o Bicarb Apr. 10, 2016 Thawed in an Fruc + 1st changing icewater bath at 41 F. No Run maple the pH motility with a Live 12/88 treesap, from 6.48 Dead stain. Tree #3. to 7.23. 2015 This appears to impairmotility. (Bad), Used 30 drops of Bicarb from a low dose insulin syringeto a 10 cc tube. 106 Apr. 3, 2016, Mar. 26, 2016 None 50 ul of Maybe;Apr. 3, 2016 Acellular, pH of Apr. 8, 2016, Semen; 50 Mistake 8 on tape,No cells seen Apr. 10, 2016 ul of BTE − made on L/D stain. Fruc +adjusting Apr. 8, 2015 Ice water thaw ½% Suc; pH up in and then palmedto warm. Plus 100 ul 2nd pH near 8 on tape. No of BTE − media cellsseen, lysed. No L/D Fruc + 1st used - stain. Run Maple using Apr. 10,2016 Ice water thaw, tree sap. Bicarb pH near 7.5 on tape. No 2015changing motility seen. Live 21/79 the pH Dead. from 6.48 to 7.23. Thisappears to impair motility. (Bad), Used 30 drops of Bicarb from a lowdose insulin syringe to a 10 cc tube. 107 Apr. 8, 2016, Mar. 26, 2016Odin None 47 ul of Maybe; Apr. 4, 2016 Ice water thaw, Apr. 4, 2016,Semen; Mistake pH 7 on tape. No motility. Apr. 8, 2016 Plus 23 ul madeLive 53/47 Dead stain. of BTE − adjusting Apr. 8, 2016 Ice water thawFruc + ½% pH up in and then palmed to warm. Suc; 2nd pH on tape was 7.2.No Plus 65 ul media motility. Live 15/85 Dead of BTE − used - stain.Fruc + 1st using Apr. 8, 2016 Ice water thaw run Maple Bicarb and thenpalmed to warm. Tree sap. changing pH 7.5 on tape. Live 13/87 2015 thepH Dead. from 6.48 to 7.23. This appears to impair motility. (Bad). Used30 drops of Bicarb from a low dose insulin syringe to a 10 cc tube. 108Apr. 4, 2016, Mar. 27, 2016 Odin None 40 ul Maybe; Apr. 4, 2016Capillary tube Apr. 10, 2016, Semen; Mistake lost from straw in thetank. Apr. 10, 2016 Plus 20 ul made Apr. 10, 2016 Ice water thaw. of BTE− adjusting No motility. pH of 7.2. No Fruc + pH up in L/D stain. ½%Suc; 2nd Apr. 10, 2016 Ice water thaw. Plus 40 ul media pH 7.2 on tape.Less than of BTE − used - 1% motile. Live 24/76 Fruc + 1st using Dead.Run Maple Bicarb Tree #3. changing 2015 the pH from 6.48 to 7.23. Thisappears to impair motility. (Bad). Used 30 drops of Bicarb from a lowdose insulin syringe to a 10 cc tube. 109 Apr. 4, 2016, Mar. 27, 2016Odin None 40 ul of Maybe; Apr. 4, 2016 Ice water thaw, Apr. 4, 2016,Semen; Mistake pH 8 on tape. No motility Apr. 10, 2016, Plus 40 ul madeand a Live 30/70 Dead Apr. 10, 2016 of BTE − adjusting stain. Fruc + pHup in Apr. 4, 2016 Ice water thaw. ½% Suc; 2nd pH on tape of 7.2. NoPlus 80 ul media motility. Live 30/70 Dead of BTE − used - stain. Fruc +1st using Apr. 10, 2016 Ice water thaw Run Maple Bicarb with pH of 7.5on tape. No Tree #3. changing motility and a Live 19/81 2015 the pH Deadstain. from 6.48 Apr. 10, 2016 Ice water thaw to 7.23. pH of 7.5. Thisappears to impair motility, (Bad). Used 30 drops of Bicarb from a lowdose insulin syringe to a 10 cc tube. 110 Apr. 3, 2016, Mar. 28, 2016Odin None 30 ul of Maybe; Apr. 3, 2016 Ice water thaw, Apr. 5, 2016,Semen; Mistake pH on tape 7.2. No Apr. 10, 2010 Plus 30 ul mademotility. Live 51/49 Dead of BTE − adjusting stain. Fruc + ½ pH up inApr. 5, 2016 Ice water thaw, % Suc; 2nd pH of 7 on tape. No Plus BTE −media motility. Live 5/95 dead Fruc + 1st used - stain. Run Maple usingApr. 10, 2016 Ice water thaw. Tree sap. Bicarb pH of 7.2 on tape. No2015 changing motility. Live 16/84 Dead the pH stain. from 6.48 to 7.23.This appears to impair motility. (Bad). Used 30 drops of Bicarb from alow dose insulin syringe to a 10 cc tube. 111 Apr. 4, 2016, Mar. 28,2016 Odin None 40 ul of Maybe; Apr. 4, 2016 Ice water thaw, Apr. 8,2016, Semen; Mistake pH of 7.2 on tape. No Apr. 8, 2016 Plus 40 ul mademotility and a Live 35/65 of BTE − adjusting Dead stain. Fruc + pH up inApr. 8, 2016 Ice water thaw ½% Suc; 2nd and then palmed to warm. Plus 60ul media pH of 7.2 on tape. Live of BTE − used - 5/95 Dead, Fruc + 1stusing Apr. 8, 2016 Less than 1% Run Maple Bicarb motility on ice waterthaw. Tree #3. changing pH 7.2 on tape. Live 7/93 2015 the pH Dead. from6.48 to 7.23. This appears to impair motility. (Bad). Used 30 drops ofBicarb from a low dose insulin syringe to a 10 cc tube. 112 Apr. 4,2016, Mar. 30, 2016 Odin None 42 ul of Yes, one Apr. 4, 2016 Ice waterthaw. Apr. 5, 2016, Semen; sample pH of 7.5 on tape. Live Apr. 5, 2016Plus 42 ul did very 25/75 Dead stain. of BTE, no well and I Apr. 5, 2015Ice water thaw fructose + wonder if and then palmed, 50% ½% Suc it frozemotile, pH 7.5 on pH tape, (pH 7.4); more Live 53/47 Dead stain. Plus 65ul slowly. Apr. 5, 2016 Ice water thaw of BTE − and then palmed to warm.Fruc + plus No motility. Live 8/92 1st Run Dead. Need slow freeze MapleTree and at least 30% sap for Sap from the cells to survive. pH Tree #3adjustments bad on (2015), (pH these samples. 7.23) 113 Apr. 4, 2016,Mar. 30, 2016 Odin None 35 ul of Maybe; Apr. 4, 2016 Ice water thaw,Apr. 5, 2016, Semen; pH no cells seen. No pH Apr. 8, 2016 Plus 35 uladjustment done. No L/D stain. of BTE − in both Apr. 5, 2016 Ice waterthaw. Fruc + ½% diluents is No motility. No pH done. Suc (pH stressingLive 19/81 Dead stain. 7.4); Plus the cells Apr. 8, 2016 Ice water thaw.35 ul of too much pH 7.3. No motility. Live BTE − Fruc + and stops 12/88Dead stain. 1st run motility. Maple Tree sap tree #3. 2015. (pH 7.23).114 Apr. 4, 2016, Apr. 2, 2016 Odin None 25 ul of Maybe; Apr. 4, 2016Ice water thaw Apr. 5, 2016, Semen; the pH with no motility. pH 8 onPlus 25 ul and tape. Live 26/73 Dead BTE − Fruc + speed of stain. ½% Sucfreezing Apr. 5, 2016 Ice water thaw (pH 7.4); need to and no motility.pH of 7.4 Plus 35 ul be on tape. Live 10/90 Dead of BTE − changed.stain. Too little sap was Fruc + 1st Slow the used and the freezing RunMaple freeze needs to be slow. pH tree sap down and needs to be lower in(pH 7.23). lower the starting extenders. 2015. No pH to other decreasecryoprotectant. cell metabolism. 115 Apr. 3, 2016, Apr. 3, 2016 OdinNone 65 ul Maybe; Apr. 3, 2016 Ice water thaw, Apr. 5, 2016, Semen; thepH pH on tape 7.4, No motility Apr. 5, 2016, Plus 65 ul and and a Live17/84 Dead Apr. 5, 2016 of BTE − speed of stain. Percentage of sap Fruc= ½% freezing needs to increase from Suc (pH need to 33%. 7.4); Plus beApr. 5, 2016 lost sample as 65 ul BTE − changed. the caulk came out onFruc + 1st Slow the thawing. Run Maple freeze Apr. 5, 2016 Ice waterthaw Tree sap. down and and then palmed to warm 2015 (pH lower thebecause caulk came out. 7.23) pH to pH 7.5 on tape. No decreasemotility. Live 8/92 Dead cell stain. metabolism. Apr. 5, 2016 Ice waterthaw and then palmed to warm. pH 7.2. No motility. Live 6/94 Dead, 116Apr. 4, 2016, Apr. 4, 2016 Odin None 40 ul of NO, NO, Apr. 4, 2016 Icewater thaw. Apr. 4, 2016, Semen; NO, pH of 7. Live 0/100 Dead Apr. 4,2016 Plus 40 ul Formula stain. No motility. of error Apr. 4, 2016 Icewater thaw. Goshawk stopping pH of 7. Live 25/75 Dead Semen motility instain. No motility. Extender = samples. Apr. 4, 2016 Ice water thaw. BTE− Fruc + The pH of 7. Live 9/91 Dead ½% addition stain. No motility. Sucw/pH of of 6.27.: glutathione Plus 80 ul and BTE − Fruc + NN- Amur Bis .. . sulfonic Maple + acid Glutathione + should NN- not have Bis . . .Sulfonic been Acid done. 117 Apr. 4, 2016, Apr. 4, 2016 Odin None 40 ulNO, NO, Apr. 4, 2016, Ice water thaw, Apr. 4, 2016, Semen; NO, pH 7, Nomotility. Live Apr. 4, 2016 Plus 40 ul Formula 28/72 Dead. Goshawkserror Apr. 4, 2016 Ice water thaw. Extender stopping pH 7. No motility.Live (pH 6.27); motility in 0/100 Dead stain. Plus 80 ul samples. Apr.4, 2016 Ice water thaw. of BTE − The pH 7, No motility. Sample Fruc +addition too small for a Live/Dead Amur of stain. 1 straw missing. Maple1st glutathione run. + and Glutathione + NN- NN Bis . . . sulfonic Bis .. . sulfonic acid acid. should not have been done. 118 Apr. 5, 2016, ( )Apr. 5, 2016 Odin None 33 ul Yes, Brix value Apr. 5, 2016 Ice waterthaw, Semen; simple 2.5 pH on tape was 7, 15-20% Plus 66 ul additionmoving initially. Live BTE − of sap 47/Dead 53. Longer Fructose +allowed acclimation may have Maple tree the cells increased survival.But sap tree #3 to longer acclimation 2015. Did survive. decreasessurvival in the not hen's seminal tubules acclimate when it does nothave an with an extender added. extender. Only acclimated semen in itsown tube and then added the sap. 119 Apr. 10, 2016, Apr. 5, 2016 OdinBack to the Yes, Yes Brix value Apr. 10, 2016, Ice water thaw, Apr. 11,2016, ( ). 2015 2.5 pH 7 on test tape, About Formulas. 5% vibrating inplace, Live The 30/Dead 70. Would likely modification have done betterif left that I above the vapors longer. made to Apr. 11, 2016 Ice waterthaw, the pH was pH 7 on tape, Some stopping vibrating in place but notmotility in moving forward, Live the cells so 31/Dead 69. It takes 90 Istarted seconds to process 1 back at sample into 4 tubes. baselineextenders. 45 ul Semen; Plus 45 ul of BTE − Fructose + ½% Sucrose (pH7.51); Plus 90 ul of BTE − Fructose + Maple Tree #3 2015 (pH 6.48) 120Apr. 10, 2016 Apr. 8, 2016 Odin Back to the Yes, the Apr. 10, 2016 Icewater thaw, 2015 original pH 7, 50% initially motile Formulas. 2015 sapto about 5% motile over The formulas about 10 minutes. Live modificationsupport 62/Dead 38. It took 90 that I the cells seconds to package the 4made to well. straws and this delay in the pH was getting it into theLN2 stopping likely allowed the osmotic motility in gradient across thecells to the cells so fade, allowing the cell to I started rehydrateprior to the back at freeze. This is conjecture, baseline but noted as aproblem in extenders. references. Need a 45 ul processing time less thanSemen, this. I stored a small then 45 ul sample of this tube in the ofBTE − fridge from 7:30 AM to Fructose + 1:30 AM and more than ½% 75%were moving forward Sucrose and straight. (not frozen). (pH 7.51), thenadded 90 ul of BTE − Fructose + Maple Tree #3 2015 (p 121 Apr. 6, 2016Odin Back to the Brix value 2015 2.5 Formulas. The modification that Imade to the pH was stopping motility in the cells so I started back atbaseline extenders. 55 ul Semen; Plus 55 ul of BTE − Fructose + ½%Sucrose (pH 7.51); Plus 100 ul of BTE − Fructose + Maple Tree #3 2015(pH 6.48) 122 Apr. 7, 2016 Odin Back to the Brix value 2015 2.5Formulas. The modification that I made to the pH was stopping motilityin the cells so I started back at baseline extenders. 123 Apr. 8, 2016Odin Back to the Brix value 2015 2.5 Formulas. The modification that Imade to the pH was stopping motility in the cells so I started back atbaseline extenders. 124 Apr. 7, 2015 Odin Back to the Brix value 20152.5 Formulas. The modification that I made to the pH was stoppingmotility in the cells so I started back at baseline extenders. 125 Apr.8, 2016 Odin Back to the Brix value 2015 2.5 Formulas. The modificationthat I made to the pH was stopping motility in the cells so I startedback at baseline extenders. 126 Apr. 10, 2016, Apr. 8, 2016 Odin Back tothe Maybe, Brix value Holder #4 trace sample Apr. 11, 2016 2015 YES 2.5because caulk came out of Formulas. capillary tube, Thawed in The IceWater, No pH, No L/D modification stain. 10% moving forward that I withgood motility.; made to Apr. 11, 2016 Ice water thaw, the pH was pH 7,30% motile on visual stopping inspection, Live 50/Dead motility in 50.the cells so I started back at baseline extenders. 40 ul Semen; Plus 40ul of BTE − Fructose + ½% Sucrose 2015 (pH 7.51); Plus 80 ul BTE −Fructose + Maple Tree sap #3 2015 (pH 6.48). 127 Apr. 10, 2016 Odin Backto the 2015 Formulas. The modifications that I made to the pH wasstopping motility in the cells so I started back at the baselineextenders. BIRCH sap STARTS HERE. 40 ul Semen, plus 40 ul of BTE −Fructose + 1st Run Alaska Birch (pH. 7.56), then added 40 ul BTE −Fructose + Maple Tree #3 2015 (pH. 6.48). 128 Apr. 10, 2016 Apr. 10,2016 Odin 40 ul Yes Apr. 10, 2016 Ice water thaw, Semen; pH 7 on tape,25% moving Plus 40 ul straight forward, Live BTE − 57/43 Dead stain.Fructose + 1st run Alaska Birch (pH. 7.56); Plus 40 ul BTE − Fructose +Maple Tree sap tree #3 2015 (pH 6.48). How sample was Holder in tank itHow storage Number frozen. pH of Diluent Diluent Type is located inStraw Type. straw performed. 4 Either flash frozen or 9.1 Diluent #1Unknown. Unknown suspended above the liquid nitrogen for 30 secondsprior to immersion. 5 Flash frozen. 9.1 Diluent #1 2 Unknown 6 Eitherflash frozen or 9.1 Diluent #1 Unknown. Natellson This straw suspendedabove the Capillary for storage liquid nitrogen for 30 tube with 2stored well seconds prior to caps. and did not immersion. explode. 7Either flash frozen or 9.1 Diluent #1 Unknown. Unknown suspended abovethe liquid nitrogen for 30 seconds prior to immersion. 8 Flash frozen.8.86 Diluent #2 2 Unknown 9 Either flash frozen or 8.86 Diluent #2Unknown. Unknown suspended above the liquid nitrogen for 30 secondsprior to immersion. 10 Flash frozen. 8.86 Diluent #2 2 11 Either flashfrozen or 8.86 Diluent #2 Unknown. Unknown suspended above the liquidnitrogen for 30 seconds prior to immersion. 12 Flash frozen. 8.86Diluent #2 2 Unknown 13 Either flash frozen or 8.86 Diluent #2 Unknown.Unknown suspended above the liquid nitrogen for 30 seconds prior toimmersion. 14 Either flash frozen or 9.08 Diluent #3 Unknown. Unknownsuspended above the liquid nitrogen for 30 seconds prior to immersion.15 Either flash frozen or Unknown None Unknown. Unknown suspended abovethe liquid nitrogen for 30 seconds prior to immersion. 16 Either flashfrozen or 9.08 Diluent #3 Unknown. Unknown suspended above the liquidnitrogen for 30 seconds prior to immersion. 17 Flash frozen. 9.08Diluent #3 3 Natellson This straw Capillary for storage tube with 2stored well caps. and did not explode. 18 Flash frozen. 9.18 Diluent #42 Natellson Capillary tube with 2 caps. 19 Either flash frozen or 7.06Trout Unknown. Natellson suspended above the Capillary liquid nitrogenfor 30 tube with 2 seconds prior to caps. immersion. 20 Either flashfrozen or 7.06 Trout Unknown. Natellson Exploded suspended above theCapillary and lost. liquid nitrogen for 30 tube with 2 seconds prior tocaps. immersion. 21 Flash frozen. 7.06 Trout 2 Natellson Capillary tubewith 2 caps. 22 Flash frozen. 7.06 Trout 5 Unknown 23 Suspended overliquid 7.16 Trout #2 Unknown. Natellson This straw nitrogen for 30seconds Capillary for storage to exposed to liquid tube with 2 storedwell nitrogen vapors and then caps. and did not flash frozen. explode.24 Suspended over liquid 7.16 Trout #2 Floating Natellson Explodednitrogen for 30 seconds on liquid Capillary on thaw to exposed to liquidnitrogen tube with 2 but did not nitrogen vapors and then and lost caps.lose the flash frozen. out of sample. holder. 25 Flash frozen. 7.16Trout #2 5 Unknown 26 Flash frozen. 7.16 Trout #2 5 Unknown 27 Flashfrozen. None 5 Unknown 28 Flash frozen. 7.16 Trout #2 5 Unknown 29Suspended over liquid 7.14 Milk #1 Unknown. Natellson This strawnitrogen for 30 seconds Capillary for storage to exposed to liquid tubewith 2 stored well nitrogen vapors and then caps. and did not flashfrozen. explode. 30 Flash frozen. 7.42 Trout #2 5 Unknown plus Sorbitoland Arabogalactin. 31 10 mm of semen to 45 Turkey 6 Commercial Explodedmm of Turkey Extender, Extender semen and lost. Acclimated in the fridgeplus DMA straw with for 30 minutes, Added 3 button ul (units) of DMA,Hung caps. in vapors for 10 minutes GREEN the placed in liquid ON TOPnitrogen. OF ALUMINUM HOLDER. 32 Semen acclimated in Turkey 6 CommercialFOUND fridge for 30 minutes, Extender semen FLOATING then DMA added,then plus (6%) straw with IN TANK hung over vapors for 10 DMA buttonMINUS minutes, then immersed caps. LABEL in liquid nitrogen. GREEN ONTOP OF ALUMINUM HOLDER. 33 Semen acclimated in Turkey 6 Commercial FOUNDfridge for 30 minutes, Extender semen FLOATING then DMA added, then plusDMA straw with IN TANK hung over vapors for 10 button MINUS minutes,then immersed caps. LABEL in liquid nitrogen. GREEN ON TOP OF ALUMINUMHOLDER. 34 Semen acclimated in Turkey 6 Commercial FOUND fridge for 30minutes, Extender semen FLOATING then DMA added, then plus DMA strawwith IN TANK hung over vapors for 10 button MINUS minutes, then immersedcaps. LABEL in liquid nitrogen. GREEN ON TOP OF ALUMINUM HOLDER. 35Semen acclimated in Turkey 6 Commercial Exploded fridge for 30 minutes,Extender semen and not then DMA added, then plus DMA straw with lost.But hung over vapors for 10 button explosion minutes, then immersedcaps. was loud in liquid nitrogen. GREEN and semen ON TOP cells likelyOF damaged ALUMINUM due to HOLDER. trauma. I am guessing that thissample went to Juniper because 4 were found floating free on top of theliquid nitrogen and not labeled. 36 See prior note Turkey 6 UnknownExtender plus DMA 37 Flash frozen. Turkey 5 Commercial Exploded Extendersemen and lost. plus DMA straw with button caps. RED ON TOP OF ALUMINUMHOLDER 38 Flash frozen. Turkey 5 Commercial Exploded Extender semen andlost. plus straw with (10%) button DMA caps. RED ON TOP OF ALUMINUMHOLDER 39 Flash frozen. Turkey 5 Commercial Exploded Extender semen andlost. plus straw with (10%) button DMA caps. RED ON TOP OF ALUMINUMHOLDER 40 Flash frozen. Turkey 5 Commercial Exploded Extender semen andlost. plus straw with The (18%) button reinforced DMA caps. RED plasticON TOP coated OF glass ALUMINUM capillary HOLDER tubes withstandexplosions. 41 SEE NOTES ON 2013 Turkey SAMPLES. Extender plus DMA 42 Infridge 10 minutes to 6.74 Turkey #2 #42, white acclimate, plus 1.5 unitsExtender straw, DMA, above vapors for plus DMA, Natellson 10 minutes,immersed 5% DMA tube, suddenly into LN2 capped on 1 end firmly andoutside end not capped. 43 Acclimated in fridge 10 6.74 Turkey #2 #43White minutes, plus 1.5 units Extender straw, DMA (5%), hung over plusDMA, standard vapors for 10 minutes 5% DMA capillary within 1 minute ofadding tube, plus DMA, dunked into LN2. two crito caps and 1 Natellsoncap. 44 Acclimated in fridge 10 6.74 Turkey #2 #44 White Straw minutes,plus 3 ul of Extender straw, exploded DMA (6%), Acclimated in plus 6%standard on thaw, fridge for 10 minutes, DMA capillary but one suspendedabove the tube plus 2 end stayed vapors 10 minutes, then critocapsclosed. dunked in LN2. and 1 Natellson cap. 45 Acclimated in fridge 106.74 Turkey #2 #45 White minutes, plus 2 ul of extender straw, DMA(10%), hung over plus 10% Standard vapors for 10 minutes, DMA capillaryplunged into LN2 tube, plus 2 caps on one end and clay on the far end.46 Acclimated in fridge (35 6.74 Turkey #2 #46 White F.), Plus 2 ul ofDMA, extender straw, tube hung over vapors for 10 plus 10% type notminutes, plunged into DMA recorded. LN2. 47 Acclimated in fridge at 356.74 Turkey #2 #47, two F. for 10 minutes, one Extender tubes, samplehung over vapors plus 6% White for 7 minutes and the DMA straw, other 10minutes, and Natellson then dunked into LN2. tube, plus clay andNatellson cap on pointed end, and Natellson cap on top end. 48Acclimated in the fridge 6.74 Turkey #2 White at 35 F., 1.5 ul of DMAextender straw, added, over vapors 10 plus 7% Natellson minutes, dunkedinto DMA. capillary LN2. tube plus clay in both ends and Natellson capon pointed end. 49 Acclimated in fridge at 6.74 Turkey #2 White 35 F.for 10 minutes, plus Extender straw, 3 ul of DMA, hung over plus 6%Natellson vapors 10 minutes, DMA tube plus plunged into LN2. clay onboth ends and rubber cap on pointed end. 50 Acclimated in fridge at 6.74Turkey #2 White 35 F. for 10 minutes, plus extender straw, 75 ul 2 ul ofDMA (7%), over plus 7% standard vapors for 10 minutes, DMA. capillarythen plunges into LN2. tube plus 1 critocap and Natellson Cap on one endand Clay on the other end. 51 Acclimated in the fridge 6.74 Turkey #2White at 35 F., 1.25 (5%) DMA extender straw, 75 ul added, hung overvapors with 5% standard for 10 minutes, plunged DMA. capillary into LN2.tube plus one end a critocap and one end a Natellson cap. 52 Acclimatedin the fridge 6.74 Turkey #2 White at 35 F., 2.7 ul of DMA extenderstraw, added, Hung over vapors with 6% Natellson 10 minutes, plungedinto DMA. Capillary LN2. tube, plus clay on both ends, and Natellson capon pointed end. 53 Acclimated in the fridge 6.74 Turkey #2 White at 35F. for 15 minutes, extender straw, 2.23 ul of DMA added, with 6%Natellson hung over vapors 10 DMA. Capillary minutes, plunged into tube,plus LN2. clay on both ends, and Natellson cap on pointed end. 54Acclimated in the fridge 6.74 Turkey #2 White at 35 F. for 15 minutes,extender straw, plus 1 ul of DMA (5%), with 6% Standard hung over vapors10 DMA. 75 ul minutes, plunged into capillary LN2. tube, clay on one endand Natellson cap and critocap on other end. 55 Acclimated in the fridge6.74 Turkey #2 White at 35 F. for 15 minutes, extender straw, added 2.5ul DMA, hung with 5% Natellson over vapors 10 minutes, DMA. tube plusplunged into LN2. clay on pointed end and Natellson caps on both ends.56 Acclimated in the fridge 6.74 Turkey #2 Red Straw, for 15 minutes,plus 1 ul extender, Natellson of DMA (5%), hung over with tube, withvapors for 10 minutes, Maple clay and plunged into LN2. Syrup, Natellsonwith 5% cap on DMA. pointed end and just clay on large end. 57Acclimated in the fridge Turkey #4 holder Red straw. The caps for 10minutes, plus 1 ul Extender in Tank 2 This was a exploded of DMA (5%),dropped by itself 75 ul mylar off of the into the LN2. coated ends andcapillary most of the tube, sample caulked on was lost. both ends, plus2 critocaps, plus a teal cap on the semen end. 58 Acclimated in thefridge Beltsville #4 Holder Red soda Sample for 15 minutes, plus 3 ulTurkey in Second straw, with explodes of DMA Extender tank. a 75 ulacross the plus .2 mg Mylar garage. Inositol to coated 10 ml ofcapillary Extender. tube, caulked on both ends, with two critocaps, andone teal cap. Plus aluminum holder. 59 Acclimated in the fridgeBeltsville #4 Holder Red soda Tube for 15 minutes, Plus 2.6 ul Turkey inthe straw, with performed of DMA and then flash Extender Second a 75 ulwell. frozen. plus .2 mg tank Mylar Inositol to coated 10 ml ofcapillary Extender. tube, caulked on both ends, with two critocaps, andone teal cap. With Aluminum holder. 60 Acclimated in the fridgeBeltsville #4 Holder Red soda Exploded for 15 minutes and then Turkey inthe straw, with but kept flash frozen Extender Second a 75 ul sample.plus .2 mg tank Mylar Inositol to coated 10 ml of capillary Extender.tube, caulked on both ends, with two critocaps, and one teal cap, WithAluminum holder. 61 Acclimated in the fridge Beltsville #3 Holder, Redsoda Straw for 15 minutes and then Turkey Tank 2 straw, with performedflash frozen Extender a 75 ul well. plus .2 mg Mylar Inositol to coated10 ml of capillary Extender. tube, caulked on both ends, with twocritocaps, and one teal cap, 62 Acclimated in the fridge Beltsville #3Holder, Red soda Straw for 15 minutes and then Turkey Tank 2 straw, withexploded flash frozen Extender a 75 ul on thaw, plus .2 mg Mylar butsample Inositol to coated was 10 ml of capillary preserved. Extender.tube, caulked on both ends, with two critocaps, and one teal cap. 63Acclimated in the fridge Beltsville #3 Holder, White soda Tube for 15minutes and then Turkey Tank 2 straw, with performed flash frozenExtender a Mylar well. plus .2 mg coated Inositol to capillary 10 ml oftube, Extender. caulked on both ends, with two critocaps, and one tealcap. 64 Acclimated in the fridge Beltsville #3 Holder, Red soda Tube for15 minutes and then Turkey Tank 2 straw, with exploded, flash frozenExtender a 75 ul Lost most plus .2 mg Mylar of the Inositol to coatedsample. 10 ml of capillary Extender. tube, caulked on both ends, withtwo critocaps, and one teal cap. 65 Acclimated in the fridge Beltsville#3 Holder, Red soda Tube for 15 minutes and then Turkey Tank 2 straw,with performed flash frozen Extender a 75 ul well. plus .2 mg MylarInositol to coated 10 ml of capillary Extender. tube, caulked on bothends, with two critocaps, and one teal cap. 66 Acclimated in the fridgeBeltsville #3 Holder, White soda Straw for 15 minutes and then TurkeyTank 2 straw, with empty on flash frozen Extender a Mylar thaw. plus .2mg coated Inositol to capillary 10 ml of tube, Extender. caulked on bothends, with two critocaps, and one teal cap, No sample in tube on thaw.67 No acclimation. Flash SAP #2 Holder Orange Trace Frozen. BeltsvilleTank 2 soda straw, sample left Turkey with 75 ul on thaw Extendercapillary due to minus the tube, plus exploding. fructose, clay bothplus ends, plus Maple critocaps. Tree sap, first run, Tree number 3. 68No acclimation, flash Likely #3 Holder, Red soda Straw frozen. same asTank 2 straw, in performed samples Aluminum well. Did above sleeve, withnot and below 75 ul mylar explode. this line. capillary tube, w/ clayand critocap with teal cap 1 end, and clay and crito cap other end. 69No acclimation was done SAP, #2 holder, Aluminum This straw and it wasflash frozen. It BTE, no Tank 2. sleeve with did well was carried to thegarage fructose, a White and did not on cold gel packs. plus soda straw,explode, Maple with 75 ul but the Tree sap, capillary large teal firstrun, tube, 1 end caps on Tree left open, the end is number 3. other endslow to capped thaw. The with clay, 1 open end critocap, 1 is key toteal cap. this success. 70 No acclimation was done SAP, #2 holder, Redsoda Straw and it was flash frozen. It BTE, no Tank 2. straw withperformed was carried to the garage fructose, holes, plus well, did oncold gel packs. plus plastic not Maple poultry explode. Tree sap, straw,with first run, a 75 ul Tree capillary number 3. tube, 1 end left open,the other end is clayed, slid into poultry straw with cotton facingdown. Straw is ventilated. 71 No acclimation was done SAP, #2 holder,Red soda and it was flash frozen. It BTE, no Tank 2. straw with wascarried to the garage fructose, holes, plus on cold gel packs. plusplastic Maple poultry Tree sap, straw, with first run, a 75 ul Treecapillary number 3. tube, 1 end left open, the other end is clayed, slidinto poultry straw with cotton facing down. 72 No acclimation was doneSAP, #2 holder, Red soda Straw and it was flash frozen. It BTE, no Tank2. straw with performed was carried to the garage fructose, holes, pluswell. on cold gel packs. plus plastic Maple poultry Tree sap, straw,with first run, a 75 ul Tree capillary number 3. tube, 1 end left open,the other end is clayed, slid into poultry straw with cotton facingdown. 73 No acclimation was done SAP, #1 holder, Pink soda Straw and itwas flash frozen. BTE, no Tank #2 straw, plus performed fructose, aplastic well. It did plus poultry not BIRCH straw, plus explode. treesap. a 75 ul Sucrose capillary is lower tube with 1 than end sealedMaple with clay, tree sap. with cotton on poultry straw facing down.Number on straw reads 67. 74 2 samples were 16% MA Holder #3 Orangeproduced due to the used as in Can #1. (#68 on volume of the sample.half of the straw) and Both were flash frozen. sample so Pink soda Nogel packs were used. the final straws (# concentration 68 on of thestraw), with MA was a plastic 8%. poultry straw inside, with a 75 ulmylar capillary tube clayed on one end, with cotton on poultry strawfacing down. 75 Separated into 3 straws 16% MA #3 Holder 3 green as itwas so large. On gel used as Tank #1. soda packs less than 2 half of thestraws, no minutes and then flash sample so holes cut in frozen. thefinal them. concentration Plastic of the poultry MA was straws with 8%.75 ul mylar capillary tubes, 1 end has clay, the cotton on the poultrystraw faces down. 76 Acclimated in the fridge 7.5 on pH Beltsville #3Holder Soda for 10 minutes, DMA tape after Turkey Tank #1. straw plusadded, used bottom of thaw. Extender, plastic fridge at 40 F. AND nopoultry TURNED DOWN THE fructose, straw, plus FRIDGE FOR THE plus ½% a75 ul FIRST TIME so it went sucrose. mylar up in temperature. THEcapillary TOP OF THE FRIDGE tube with WAS AT 35 F. AND THE clay on 1BOTTOM OF THE end. With FRIDGE WAS AT 40 F. cotton on BEFORE I TURNED ITpoultry DOWN. It now sits at 42 straw at the bottom where I facing amnow holding the down. Not samples so it is 9 ventilated. degrees warmedfor Blue in processing. The color. samples done from here out are doneat a warmer temperature. 77 Flash frozen after 10 7.5 pH on BTE, noHolder #3 3 soda minutes of acclimation at pH tape fructose, in Can #1.straws, 41 F. after plus ½% light green, thaw. sucrose, dark green, andDMA orange, with a poultry straw inside that and a 75 ul mylar tubeinside of that. Green straw was not ventilated and the Orange straw wasnot ventilated. 78 Flash frozen after 10 BTE, no Holder #3 2 sodaStorage minutes of acclimation at fructose, in Can #2. straws, with a 41F. plus Birch both pink, Ventilated Tree sap. both soda straw,ventilated, poultry with poultry straw straw and a inside that 75 ulmylar [Poultry capillary straw tube inside crimped on that. Clay top endon one end and cotton leaving it on the open on bottom top. end], with a75 ul mylar coated capillary tube, clay on one end only with the otherend left open to LN2; Performed the BEST Keep the material open so theydo not explode. All samples stored this way from this point on. 79Sample was not Birch tree Holder #3, 2 Soda Well, no acclimated in thefridge. sap in Tank #2. straws, one problems. It was mixed, placed inBTE, No yellow and tubes, and then put fructose one green, between gelpacks from both the fridge, carried out to ventilated the LN2 can, andflash with holes; frozen. with a poultry straw, cotton facing down,crimped on top end; with 75 ul capillary tubes, clayed on one end. 80Acclimated in the fridge Maple Holder #3, 2 soda Well, no for 10 minutesin tree sap Tank #2. straws, one problems. separate tubes, then in BTE,orange and combined and packaged, no one pink, then flash frozenfructose. both ventilated, with poultry straw inside, with 75 ul mylarcoated capillary tube inside. 81 Acclimated for 10 Maple Holder #3, 2Soda Well, no minutes in the fridge at tree sap Tank #2. straws thatproblems. 41 F., and then flash in BTE, were frozen no ventilated,fructose. with poultry straw inside, cotton end down; 75 ul mylarcapillary tube, clay on one end 82 Semen and BTE − Fruc, + pH on pHBirch tree 2 straws 4 green Does not ½% sucrose tape after sap in inHolder soda explode acclimated in separate thaw was BTE, No #6 tank 1,straws that but thaws tube from Birch sap BTE, 7.0, 7.2, fructose and 2were too slowly. at 42 F. in fridge for 10 7.0, 7.5. straws inventilated, minutes, combined, then Holder #5 75 ul mylar packaged in 4tubes, 75 tank 1. capillary ul capillary tube, caulked tube, inside oneend; placed in poultry poultry straw, cotton straw, down; insideventilated inside soda straw. 4 straws ventilated made. All green sodasoda straw. straws. 83 Semen and BTE − Fruc, + pH on pH Maple 2 straws 3yellow Good ½% sucrose tape after tree sap in Holder soda acclimated inseparate thaw was in BTE, #6 tank 1, straws that tube from Maple treesap 7.5-8, 7.0, 7.0 no and 1 were BTE, at 42 F. in fridge for fructose.straws in ventilated, 10 minutes, combined, Holder #5 75 ul mylar thenpackaged in 4 tank 1. capillary tubes, 75 ul capillary tube, insidetube, caulked one end; poultry placed in poultry straw, straw, cottondown; inside inside ventilated soda straw. 3 ventilated straws made. 3Yellow soda straw. straws. 84 Semen and BTE − Fruc, + Mar. 1, 2016 Maple2 straws 3 pink soda Tubes ½% sucrose pH on tree sap in Holder strawsthat store well acclimated in separate tape of 7. in BTE, #6 tank 1,were but it is tube from Maple tree sap Mar. 6, 2016 no and 1ventilated, best to use BTE, at 44 F. in fridge for pH on fructose.straws in 75 ul mylar the soda 10 minutes, combined, tape of 7.5. Holder#5 capillary straw and then packaged in 3 tank 1. tube, inside the mylartubes, 75 ul capillary poultry capillary tube, caulked one end; straw,tube only. placed in poultry straw, inside cotton down; insideventilated ventilated soda straw. 3 soda straw. straws made. 3 Pink Onestraws. sample was a capillary tube and soda straw only. 85 Semen andBTE − Fruc, + pH on pH Maple 2 straws 3 Yellow ½% sucrose tape was treesap in Holder soda acclimated in separate 7.0, pH on in BTE, #6 tank 1,straws that tube from Maple tree sap pH tape no and 1 were BTE, at 44 F.in fridge for was 7.0 fructose/ straws in ventilated, 10 minutes,combined, with 10% Holder #5 75 ul mylar then packaged in 3 Yolk.tank 1. capillary tubes, 75 ul capillary tube, inside tube, caulked oneend; poultry placed in poultry straw, straw, cotton down; inside insideventilated soda straw. 3 ventilated straws made. 3 Yellow soda straw.straws. 10% Yolk Added to both mixes. 86 All volumes added Maple Holder#5, 1 pink soda together, No acclimation. tree sap Tank 1. straw thatFlash frozen. 10% Yolk in BTE, was Added. no ventilated, fructose/ 75 ulmylar with 10% capillary Yolk. tube, inside a poultry straw, inside thesoda straw. 87 Acclimated for 10 pH of 7 on Maple Holder #6, UnknownStores minutes in the fridge at pH tape tree sap in can #1. color ofwell, but 43 F., and then flash after in BTE no soda straw. thaws toofrozen. 10% Yolk thaw. fructose/ Was a Pink slow. added. with yolk.ventilated soda straw. 88 Acclimated for 10 2016 pH Maple Holder #6, Twopink Straws minutes in the fridge at on pH tree sap in can #1 soda workwell 43 F., and then flash tape 7, pH in BTE no has 2 pink straws, butinsulate frozen. 10% Yolk on pH fructose/ straws, ventilated, too wellon added. tape of 7 with yolk. and with a 75 ul thawing. Holder #5,mylar in can #1 capillary has 1 pink tube inside straw. a poultry straw.89 Acclimated for 10 2016 pH Birch tree Holder #6 Two Does not minutesin the fridge at on tape sap in has 1 orange explode 43 F., and thenflash of 7.0 BTE, No orange soda but thaws frozen. 10% Yolk fructose,soda straws, too slowly. added. Plus straw, and ventilated, YOLK Holder#5 with 75 ul has 1 mylar orange capillary soda tubes straw. insidepoultry straws. 90 Acclimated for 10 pH on Birch tree Holder #6 ThreeDoes not minutes in the fridge at tape of sap in has 2 green sodaexplode 43 F., and then flash 7.0, 7.0, BTE, no green straws, but thawsfrozen. 10% Yolk 7.0. fructose soda ventilated, too slowly. added. PlusYolk. straws with 75 ul and mylar Holder #5 capillary has 1 tubes greeninside soda poultry straw. straws. 91 Temperature dropped pH on MA onlyHolder #4, 2 soda only by using gel packs. tape after Tank 1. 2 straws,one thaw was straws, green and 7.5, 7.5 one is one deep green and blue.the other Ventilated deep with 75 ul purple. mylar capillary tube insidea poultry straw. 92 Temperature dropped MA only Holder 3, 1 green Storesonly by using gel packs. Tank 1, 1 soda straw, well, but greenventilated. thaws too soda With 75 ul slow. straw. mylar capillary tubeinside a poultry straw. 93 Chilled at 45 F. for 10 pH on MA only Holder4, 3 green minutes and then flash tape after Tank 1, 3 soda frozen. thawwas green straws, 7.5, 7.5, 7.5. soda ventilated, straws all with 75 ulhere. mylar capillary tube inside a poultry straw. 94 Chilled at 45 F.for 10 DMA Holder #5, 1 orange minutes and then flash Tank 1. soda strawfrozen. with 2 poultry straws inside of it because I ran out of staplesand had 1 soda straw left with a staple in it, marked with # 89 on it.95 It was mixed at room 2016 pH 7 Purdy Holder 5 2 orange Ventilatedtemperature with no 10% and 6, soda soda straw acclimation and thenMaple Tank 1. straws with with poultry flash frozen. Lack of Tree Sap,75 ul mylar straw and acclimation reduces cell 326 capillary mylarsurvival. mOsm. tubes capillary inside tube inside poultry that thawsstraws. too slowly. 96 It was mixed at room Purdy Holder # 1 lighttemperature with no 10% 5, Tank 1, yellow soda acclimation and thenMaple Light straw with flash frozen. Tree Sap, yellow 75 ul mylar 326soda capillary mOsm. straw. tube inside a poultry straw. 97 It was mixedat room pH of 7 on Purdy Holder # 1 pink soda Stores temperature,sandwiched pH tape 10% 6, Tank 1. straw that well, but between gel packsat 43 after Maple Pink soda was thaws too F., and then flash frozen.thaw. Tree Sap, straw. ventilated, slow. 326 75 ul mylar mOsm. capillarytube, inside a poultry straw, inside the soda straw. 98 Purdy formulasbegin with # 95. 99 It was mixed at room Feb. 24, 2016 Purdy 20% Holder#4 3 pink soda temperature, sandwiched Pink Maple has 2 pink straws thatbetween gel packs at 43 straw, no Tree Sap, soda are F., and then flashfrozen. pH done, 308 straws ventilated, Second mOsm. and 75 ul mylarstraw pH Holder #5 capillary of 7.5, has 1 pink tube, inside Last strawsoda a poultry no pH straws. straw, done. inside the soda straw. 100 Itwas mixed at room Mar. 6, 2016 Purdy 20% Three 4 greens Good,temperature, sandwiched pH 7 on Maple green soda stores well between gelpacks at 43 tape after Tree Sap, soda straws that but thawed F., andthen flash frozen. thaw. 308 straws in are too slow. Mar. 6, 2016 mOsm.Holder #6, ventilated, pH of 7 on 1 straw in 75 ul mylar tape afterholder #5. capillary thaw. tube, inside a poultry straw, inside the sodastraw. 101 It was mixed at room Mar. 2, 2016 Purdy Two 2 Yellow Sodastraw temperature, sandwiched pH of 7 on 10% yellow soda did not betweengel packs at 43 pH tape Maple straws, straws that have F. for 2 minutes,and then after tree sap one in are capillary flash frozen. thaw. holder#6, ventilated, tube in it, and one 75 ul mylar Lost in the in holdercapillary tank. #5. tube, inside a poultry straw, inside a soda straw.102 It was mixed at room Mar. 2, 2016 Purdy 5% 3 orange 3 orange Good,temperature, sandwiched pH of 7; Maple soda soda stores well between gelpacks at 43 Mar. 6, 2016 tree sap. straws, 2 straws that but thawed F.for 2 minutes, and then pH of 7. in holder are too slow. flash frozen.No #6, and 1 ventilated, acclimation. in holder 75 ul #5 capillary tubeinside a poultry straw. 103 It was mixed at room 7.5 and 7, Purdy 4green 4 green Good temperature, with no 7, 7. 10% soda soda acclimation,and then Maple straws, 2 straws that flash frozen. tree sap, straws inwere plus 5% holder #5 ventilated, (13 ul) of and 2 75 ul mylar DMA.straws in capillary holder #6. tube, inside poultry straw, insideventilated soda straw. 104 It was mixed at room 7 Purdy Yellow YellowGood temperature, with no 10% straw in soda straw acclimation, and thenMaple holder #5 that was flash frozen. tree sap ventilated, plus 75 ulmylar Arabogalactin capillary and tube, inside then 12% poultry MA.straw, inside ventilated soda straw. 105 Chilled 15 minutes and 7.51 and7.23. 2015 2 straws Mylar Stores well then second diluent (adjustedextenders in Holder capillary and thaws added. Packaged quickly wrongthat have #6, 1 tube, well. and then flash frozen. and bad sap in strawin caulked on for them. Holder #5 one end, motility). Second inside adiluent small had pH ventilated adjusted soda straw. up with bicarb. 106Acclimated in the fridge 7.51 and 7.23. 2015 2 straws Mylar Stores wellat 42 F. for 15 minutes (adjusted extenders in Holder capillary andthaws and then flash frozen. wrong that have #6 and 1 tube, well. andbad sap in straw in caulked on for them. Holder #5. one end, motility).Second inside a diluent small had pH ventilated adjusted soda straw. upwith bicarb. 107 Acclimated 16 minutes at pH 7.51 2015 2 straws MylarStores well 42 F. and then packages then extenders in Holder capillaryand thaws into 3 straws and flash pH 7.23 that have #6 and 1 tube, well.frozen. (adjusted sap in straw in caulked on wrong them. Holder #5. oneend, and bad Second inside a for diluent small motility). had pHventilated adjusted soda straw. up with bicarb. 108 Acclimated 16minutes at pH 7.51 2015 2 straws Mylar Stores well 42 F. and thenpackages then extenders in Holder capillary and thaws into 3 straws andflash pH 7.23 that have #6 and 1 tube well. frozen. (adjusted sap instraw in caulked wrong them. Holder #5. one end and bad Second with fordiluent ventilated motility). had pH soda straw. adjusted up withbicarb. 109 Acclimated 16 minutes at pH of 7.51 2015 2 straws MylarStores well 42 F. and then packages then pH extenders in Holdercapillary and thaws into 4 straws and flash of 7.23 that have #6 and 2tube well. frozen. (adjusted sap in straws in caulked wrong them. Holder#5. one end and bad Second with for diluent ventilated motility). had pHsoda straw. adjusted up with bicarb. 110 Acclimated 16 minutes in pH of7.51 2015 2 straws Mylar Stores well the fridge at 42 F. and then pHextenders in Holder capillary and thaws then flash frozen. of 7.23 thathave #6 and 1 tube well. (adjusted sap in straw in caulked wrong them.Holder #5. one end and bad Second with for diluent ventilated motility).had pH soda straw. adjusted up with bicarb. 111 Acclimated for 18 pH of7.51 2015 2 straws Mylar Stores well minutes at 42 F. in the then pHextenders are in capillary and thaws fridge and then flash of 7.23 thathave Holder #6 tube well. frozen. (adjusted sap in and 1 caulked wrongthem. straw is in one end and bad Second Holder #5. with for diluentventilated motility). had pH soda straw. adjusted up with bicarb. 112Acclimated on gel packs 7.4 then BTE 1 straw in Mylar Caulk at 42 F. for16 minutes. 7.23 both minus holder #5 capillary tends to Then flashfrozen. One 2015 diluents fructose, and 2 tube come out sample gotcaught on the with sap plus ½% straws in caulked on thaw holder andstayed above that both sucrose holder #4. one end due to LN2 the LN2 andwas not had pH with pH with pressure flash frozen. The other adjustmentsadjusted ventilated inside the samples in the group that with sodastraw. capillary were flash frozen and went bad. bicarb tube. died. from7.51 to 7.4.; Then added BTE − Fructose + Maple Tree Sap tree #3 with pHadjusted with bicarb to 7.23. 113 Acclimated for 16 7.4 then BTE 1 strawin Mylar Stores well minutes in the fridge at 7.23 both minus holder #5capillary and thaws 42 F. and then flash 2015 diluents fructose, and 2tube well. frozen. with sap plus ½% straws in caulked that both holder#4. one end had pH sucrose with adjustments with pH ventilated thatadjusted soda straw. went bad. with bicarb from 7.51 to 7.4.; Then addedBTE − Fructose + Maple Tree Sap tree #3 with pH adjusted with bicarb to7.23. 114 Acclimated for 16 7.4 then BTE 1 tube in Mylar minutes in thefridge at 7.23 both minus Holder #5 capillary 42 F. and then flash 2015diluents fructose, and 1 tube frozen. with sap plus ½% tube in caulkedthat both sucrose Holder #4. one end had pH with pH with adjustmentsadjusted ventilated that with soda straw. went bad. bicarb from 7.51 to7.4.; Then added BTE − Fructose + Maple Tree Sap tree #3 with pHadjusted with bicarb to 7.23. 115 Acclimated 20 minutes and then flashfrozen. 116 Acclimated 15 minutes pH 6.27 Amur 3 straws Mylar Storeswell and then flash frozen. then Maple + in Holder capillary and thawspH 6.74. BTE − #3. tube well. Fruc with caulked adjusted one end pH of6.74 with with ventilated Glutathione + soda straw. NN- Bis . . .Sulfonic Acid. 117 Acclimated 15 minutes pH 6.27 Amur 4 straws MylarStores well and then flash frozen. then Maple + in Holder capillary andthaws pH 6.74. BTE − #3. tube well. Fruc with caulked adjusted one endpH of 6.74 with with ventilated Glutathione + soda straw. NN- Bis . . .Sulfonic Acid. 118 Acclimated semen in its pH 6.48 BTE − 2 straws Mylarown tube for 5 minutes Fructose + in holder capillary and then added inthe Maple #3. tube + Maple tree sap tree #3 Tree #3 Critocap + (2015).Then slowly sap, Blue cap + lowered into LN2. 2015. Ventilated (originalsoda straw. 6.48 pH from 2015) 119 Put in holder #5 above 7.51. then6.48 BTE − #5 Mylar Caulk liquid nitrogen vapors for Fructose +capillary tends to 10 seconds and then ½% tube, come out flash frozewith slow Sucrose caulked on on thaw immersion. (pH 7.51), one end, dueto LN2 then BTE − inside a pressure Fructose + small inside the Mapleventilated capillary Tree #3 soda straw. tube. sap, 2015. (pH 6.48). 120Acclimated 15 minutes at 7.51 then 6.48 BTE − 4 straws Mylar Caulk 42 F.then hung over the Fructose + in Holder capillary tends to vapors 15seconds and ½% #3. tube, come out then slowly lowered into Sucrosecaulked on on thaw LN2. (pH 7.51); one end, due to LN2 then BTE − insidea pressure Fructose + small inside the Maple ventilated capillary Tree#3 soda straw. tube. sap, 2015. (pH 6.48). 121 Acclimated 15 minutes at7.51 then 6.48 BTE − 2 straw in Natellson 42 F. then hung over theFructose + Holder #3. Capillary vapors 15 seconds and ½% tube with 1then slowly lowered into Sucrose cap in a LN2. (pH 7.51); large then BTE− ventilated Fructose soda straw. plus (Orange). Maple Tree sap #3 2015(pH. 6.48) 122 Acclimated 15 minutes at pH 7.51 BTE − 1 straw inNatellson 42 F. then hung over the then Fruc + Holder #3. capillaryvapors 15 seconds and pH 6.48. ½% Suc tube + cap + then slowly loweredinto (pH 7.51); Large LN2. then BTE − ventilated Fruc + soda straw 1stRun (Green). Maple tree sap. 2015 123 124 125 126 Acclimated for 15 pH7.51 BTE − Three Mylar Caulk minutes at 42 F. in the then Fructose +straws in capillary tends to fridge, and then flash pH 6.48. ½% Holder#5, tube come out frozen in LN2. Sucrose 1 Straw in caulked on thaw 2015Holder #4. one end due to LN2 (pH 7.51); with pressure then ventilatedinside the added soda straw. capillary BTE − tube. Fructose + Maple TreeSap tree #3 2015 (pH 6.48) 127 pH 7.56 #4 Mylar then capillary pH 6.48tube caulked one end with ventilated soda straw. 128 Acclimated for 15pH 7.56 BTE − 3 straws Mylar Caulk minutes then suspended then Fructosein Holder capillary tends to over LN2 vapors for 15 pH 6.48 plus 1st #4.tube come out seconds and then slowly run caulked on thaw lowered intoLN2. Alaska one end due to LN2 Birch with pressure (pH 7.56) ventilatedinside the and then soda straw. capillary BTE − tube. Fructose + MapleTree #3 2015 (pH 6.48)

1-12. (canceled)
 13. A method of cryogenically preserving spermcomprising: a. combining sperm to be cryogenically preserved and acomposition that comprises (1) a cryoprotectant, comprising one or moretree saps; and (2) an extender medium to produce a sperm/mediumcombination; and b. subjecting the combination to conditions that resultin cryopreservation of sperm, thereby producing a cryopreservedcombination that comprises cryopreserved sperm.
 14. The method of claim13 wherein the cryopreserved sperm of step (b) demonstrates survival atgreater than 50% after thawing as demonstrated by Eosin/Nigrosinlive/dead staining.
 15. The method of claim 14 wherein the survival isat least 73%.
 16. The method of claim 13 wherein the cryopreserved spermof step (b) demonstrates motility at greater than 30% after thawing. 17.The method of claim 13 wherein the cryopreserved sperm of step (b)demonstrates motility of at least 50% after thawing.
 18. The method ofclaim 13 wherein the one of more tree saps is the only cryoprotectant.19. The method of claim 13 wherein an additional cryoprotectant isadded.
 20. The method of claim 13 wherein the sperm is avian sperm. 21.The method of claim 13 wherein the sperm is derived from the Northerngoshawk (Accipiter gentilis).
 22. The method of claim 13 wherein thesperm is derived from a non-human mammal.
 23. The method of claim 13wherein the sperm is derived from a species selected from the groupconsisting of canine, avian, cattle, porcine and equine.
 24. The methodof claim 13 wherein the sap is either maple tree sap or birch tree sap.25. The method of claim 13 wherein the sap is a first run sap.
 26. Themethod of claim 13 wherein the extender medium does not containfructose.
 27. The method of claim 13 wherein the method comprises theadditional step of subjecting the combination to a temperature between−80° F. and −198° F. for a period of at least one day.
 28. Thecryopreserved combination resulting from the method of claim
 13. 29. Thecryopreserved combination resulting from the method of claim
 14. 30. Thecryopreserved combination resulting from the method of claim
 16. 31. Amethod of fertilizing an egg cell comprising the step of thawing thestep (b) product and introducing the combination to an unfertilized eggcell, wherein the egg becomes fertilized.
 32. The method of claim 30,wherein the egg is an avian egg.
 33. The method of claim 31, wherein theegg is a mammalian egg.